Conjugation chemistry is a major determinant of the structural homogeneity, stability, pharmacokinetics, manufacturability, and therapeutic index of antibody-drug conjugates (ADCs). Conventional conjugation through solvent-accessible lysine residues or cysteines generated by reduction of interchain disulfide bonds has enabled the development of many clinically successful ADCs. However, these approaches can produce mixtures that differ in drug-to-antibody ratio (DAR) and attachment site, complicating product characterization and, in some settings, contributing to variable stability, clearance, efficacy, and off-target toxicity. This review examines how advances in conjugation technology address these limitations and how the choice of conjugation strategy influences chemistry, manufacturing, and controls (CMC), preclinical performance, and clinical translation. For clarity, the principal conjugation approaches are organized into five broadly defined and partially overlapping categories: nonspecific conjugation; enzyme-mediated site-specific conjugation; click chemistry and bioorthogonal conjugation, including chemoenzymatic glycan remodeling; photocatalytic site-selective conjugation; and conjugation based on genetically encoded noncanonical amino acids. This framework is intended to facilitate comparison rather than to represent mutually exclusive technological generations. For each category, we compare the origin and accessibility of the conjugation site, reaction mechanism, operating conditions, achievable drug-to-antibody ratio (DAR) distribution, requirement for antibody engineering, product homogeneity, linker stability, scalability, and stage of development. Enzyme-mediated approaches involving sortase A, microbial transglutaminase, formylglycine-generating enzyme, or ligase-dependent conjugation enable controlled modification at defined residues or engineered recognition motifs. Click and bioorthogonal reactions, particularly strain-promoted azide-alkyne cycloaddition, permit rapid and selective coupling under mild conditions and can be combined with glycan remodeling or orthogonal reactive handles to generate homogeneous, dual-payload, or multi-payload ADCs. Photocatalytic site-selective approaches provide temporal control over bioconjugation reactions but remain predominantly at the methodological or preclinical stage. Genetic code expansion enables the precise incorporation of reactive noncanonical amino acids, although limited expression efficiency, manufacturing complexity, and production cost remain important barriers to broader application. Comparative analysis indicates that no single conjugation platform is optimal for every ADC. Greater site control and a narrower DAR distribution can improve product consistency and may reduce the rapid clearance or aggregation associated with some highly loaded species. These advantages, however, must be balanced against antibody engineering requirements, enzyme or reagent supply, reaction yield, purification burden, analytical complexity, scale-up feasibility, and regulatory expectations. Moreover, clinical performance cannot be attributed to conjugation chemistry alone. Target biology, antigen density and heterogeneity, antibody properties, linker cleavage, payload potency and permeability, DAR, dosing regimen, and patient selection jointly determine efficacy and safety. Advantages observed in platform studies should therefore not be assumed to confer clinical benefit without appropriate comparative evidence. Representative approved and clinical-stage ADCs are considered to illustrate both the contribution and the limits of conjugation strategy in clinical development. Persistent translational challenges include premature payload release, off-target uptake, dose-limiting hematologic or organ toxicities, tumor heterogeneity, impaired internalization and lysosomal processing, drug-efflux mechanisms, and resistance to payload-induced cell death. These issues emphasize the need to integrate conjugation-site selection with linker and payload design rather than optimize each component in isolation. Future development is expected to combine site-controlled conjugation with hydrophilic linkers, dual-payload or bispecific architectures, conditionally activated release mechanisms, and biomarker-guided patient stratification. Expansion into autoimmune and infectious diseases will place additional demands on selectivity, tolerability, and manufacturing consistency. Overall, rational selection of conjugation chemistry, supported by rigorous analytical characterization and disease-specific pharmacology, should help translate structurally defined ADCs into products with more predictable quality and wider therapeutic windows.