Homocysteine exists in human plasma as various (mixed) disulfides. Most plasma homocysteine (about 70%) is protein bound, probably via a disulfide bond to albumin, whereas homocysteine-cysteine mixed disulfide is the predominating form in the free fraction. We here present a method for the determination of total homocysteine, which includes both fractions. Plasma was initially treated with sodium borohydride to reduce the disulfide bonds, and the liberated thiols were derivatized with monobromobimane. The derivatized sample, still containing the plasma proteins, was injected onto a strong cation-exchange column, from which the homocysteine derivative was directed by column switching into a cyclohexyl silica (CH) column. The homocysteine derivative was top-concentrated on the CH column, then rapidly eluted with a steep gradient of methanol. Both the derivatization procedure and chromatography were performed with a combined sample processor and sample injector from Gilson (Model 232-401). Within-run and between-run precision (CV) was less than 4%, and the detection limit of 0.2 pmol was sufficiently low for monitoring homocysteine in plasma. We verified the assay against two established manual methods for the determination of total homocysteine in plasma. This, the first fully automated assay for total plasma homocysteine, allows the unattended analysis of 70 samples per 24 h.