Tyrosine phosphorylation is a fundamental mechanism for regulating the functions of numerous proteins in eukaryotic cells. It has been known for some time that several members of the Rab GTPase family can undergo phosphorylation on serine or threonine residues, but the potential for tyrosine phosphorylation has been appreciated only recently, based on a single example-Rab24. Herein we describe a series of straightforward methods to facilitate an initial assessment of the potential for tyrosine phosphorylation of epitope-tagged Rab proteins transiently expressed in mammalian cells. The approach takes advantage of the availability of highly specific monoclonal antibodies against phosphotyrosine and specific chemical inhibitors for tyrosine kinases. We also describe the use of site-directed mutagenesis to identify tyrosine residues that may be targets for phosphorylation, and we discuss the possible relevance of this modification for regulating Rab function.