Tyrosine sulfation is a post-translational modification (PTM) where a sulfate group is added to a tyrosine moiety. This PTM is responsible for strengthening interaction between proteins. One of the drawbacks of studying this PTM is the lack of an antibody that can detect all tyrosine-sulfated proteins. In addition, due to the labile nature of the tyrosine sulfate, other techniques such as mass spectrometry cannot be used to study this PTM unless special modification procedures are used. This requires considerable skill and knowledge of mass spectrometry. This unit describes an in vitro technique that can be used to study tyrosine-sulfated proteins by radiolabeling the recombinant protein. The protein is then subject to barium hydroxide hydrolysis and thin-layer electrophoresis (TLE). Co-localization of radioactive tyrosine-sulfate with nonradioactive tyrosine sulfate standard spiked in before TLE analysis identifies a protein as tyrosine-sulfated protein. The advantage of this technique is that, it identifies all tyrosine-sulfated proteins without any bias and is the only technique that identifies the tyrosine sulfate residues in the protein.