Recombinant Factor VIII (rFVIII) is involved in the cascade of biochemical reactions leading to blood coagulation and is used for the treatment of haemophilia A. Plasma-derived FVIII (pdFVIII) has been reported to be post-translationally modified by sulfation of tyrosine residues at positions 346, 1664, 1680, 718, 719 and 723. This report describes the quantitation of tyrosine sulfate residues in BHK-derived, human rFVIII by amino acid composition analysis and the identification of their positions in the polypeptide sequence using a combination of liquid chromatography and electrospray ionization mass spectrometry in the analysis of proteolytic digests of the protein.