Homocysteine is a bioactive compound involved in many impotant processes of clinical significance. High sensitivity methods associated with preparation of oxidation-resistant derivatives are of demand for determination of different homocysteine fractions. In this work a derivatization agent N-ethylmaleinimide (NEM) was proposed by us for detection of reduced homocysteine fraction with HPLC-MS. Linearity, detection limit, and precision were 25-1500 nM, 10 nM, and 95-105% respectively, with reproducibility within 12%.