Small proteins (< 100 amino acids) are prevalent in all three domains of life. Earlier studies have been focusing on a limited number of small protein families in specific organisms and developing genome-wide algorithms to identify short open-reading-frames or sORFs. Here the in silico analyses on small proteins (SPs) include both known SPs and genes with sORFs. RefSeq proteins that shorter than 100 amino acids in length are defined as SPs and are grouped according to their sequence conservation within lineages of eukaryotes, vertebrates, and mammals. Biological roles of the grouped SPs are found basically performing lineage-specific functions. Tissue-specificity of human SPs are also investigated and showed that a majority of the human-specific SPs are tissue-specific and that most of the human SPs originated after the split of vertebrates and invertebrates are mostly universally expressed. In addition, the results indicated that some of the eukaryotic SPs perform lineage-specific functions and they evolve and express in certain unique ways.