The O-sulfation of tyrosine residues of plasma membrane and secretory proteins that transit through the secretory pathway of eukaryotic cells is a widespread post-translational modification. This enzymatic reaction is catalyzed by trans-Golgi-associated tyrosylprotein sulfotransferases, which recognize tyrosine residues located in a specific acidic amino acid sequence. Tyrosine sulfation promotes extracellular protein-protein interactions involved in diverse biological processes, ranging from the receptor binding of regulatory peptides to the interaction of viral envelope proteins with the cell surface. This unit outlines procedures to determine whether a protein of interest contains sulfated tyrosine residues, using methods based on labeling proteins with inorganic [35S]-sulfate, alkaline hydrolysis, and one-dimensional thin-layer electrophoresis.