The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H14CO3- into synthetic peptides and subsequent quantification using liquid scintillation counting. We present a nonradioactive assay using a fluorescein isothiocyanate-labeled short peptide that can be readily detected in its unmodified and γ-glutamyl carboxylated form by reversed-phase HPLC. This method offers a convenient alternative to the established radioactive labeling techniques.