A simple methodology for the identification of hemostatic proteins that are subjected to posttranslational tyrosine sulfation was developed. The procedure involves sequence analysis of members of the three hemostatic pathways using the Sulfinator prediction algorithm, followed by [(35)S]sulfate labeling of cultured HepG2 human hepatoma cells, immunoprecipitation of targeted [(35)S]sulfate-labeled hemostatic proteins, and tyrosine O-[(35)S]sulfate analysis of immunoprecipitated proteins. Three new tyrosine-sulfated hemostatic proteins-protein S, prekallikrein, and plasminogen-were identified. Such a target-specific approach will allow investigation of tyrosine-sulfated proteins of other biochemical/physiological pathways/processes and contribute to a better understanding of the functional role of posttranslational tyrosine sulfation.