Anoikis is a type of apoptosis that results from cell detachment from the extracellular matrix. Resistance to anoikis may allow survival of cancer cells during metastasis. It has been found that PI3K-PKB/Akt and MAPK pathways confer anoikis resistance to cancer cells. However, the tyrosine kinase pathways upstream of PI3K-PKB/Akt and MAPK have not been adequately explored. In an attempt to identify specific phosphotyrosine-containing proteins and potential tyrosine kinase pathways involved in anoikis resistance, a functional screening method based on the specific interaction between src homologue 2 (SH2) domains and phosphotyrosine (p-Tyr) containing proteins was designed. Cell detachment rendered normal MDCK cells to undergo anoikis. However, the survival and proliferation of tumor cells was anchorage-independent. Consistent with this phenomenon, cell detachment induced rapid decrease in SH2s-binding tyrosine phosphorylated proteins in MDCK cells, while tyrosine phosphorylation of SH2-binding proteins in tumor cells was anchorage-independent. It was also found that tyrosine phosphorylation levels of Abl SH2-associated proteins decreased in detached MDCK cells. However, the tyrosine phosphorylation levels of Abl SH2-associated proteins in H460 lung tumor cells increased after a transient decrease, and the levels increased in H1792 lung tumor cells upon detachment. Using this functional screening method, some of the Fyn SH2 and Crk SH2-binding proteins were identified as FAK and p130Cas respectively, which are critical in mediating cell-matrix interactions. The present data suggest that multiple phosphotyrosine-containing proteins and potential tyrosine kinase pathways may act to support the anoikis resistance of tumor cells. The SH2-domain screening method may be an efficient way to explore anoikis-resistance-related phosphotyrosine-containing proteins and potential tyrosine kinase pathways in tumor cells.