Dihydroergocristine (DHEC) is a semi-synthetic drug mainly used for age-related cognitive impairment. In this study, its major metabolite 8-hydroxy-dihydroergocristine (8-OH-DHEC) was produced in incubates of a bovine liver preparation using dihydroergocristine mesylate (DHECM) as substrate. Purification was achieved by flash silica gel column and reverse phase liquid chromatographies, and identification was based on accurate molecular mass measurements, mass fragmentation spectra and NMR ( 1H/13C) chemical shifts. By using the substance produced in vitro, a fast, sensitive, specific and robust LC/MS/MS method for the simultaneous determination of DHEC and its major metabolite in human plasma was developed and validated. Bromocriptine was used as internal standard and limits of quantification for DHEC and 8-OH-DHEC were 10 pg/ml and 20 pg/ml, respectively. Pharmacokinetic parameters were investigated on 12 male healthy volunteers to whom a single dose of 18 mg DHECM was administered in tablets (Iskevert®). The peak of DHEC was 0.28 ± 0.22 μg/l, the tmax 0.46 ± 0.26 h, the AUClast 0.39 ± 0.41 µg/l.h and the terminal elimination half-life 3.50 ± 2.27 h. The peak of 8-OH-DHEC was 5.63 ± 3.34 µg/l, the tmax 1.04 ± 0.66 h, the AUClast 13.36 ± 5.82 μg/l.h and the terminal elimination half-life 3.90 ± 1.07 h. Dosing of 18 mg DHECM was well tolerated, causing no adverse events.