Protein tyrosine phosphorylation is a reversible post-translational modification that is essential for life in eukaryotic cells. The combinatorial action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs) determines the net level of cellular tyrosine phosphorylation. This unit discusses methods to determine the level of protein tyrosine phosphatase activity and methods for discovering novel substrates for protein tyrosine phosphatases.