This paper describes for the first time the enantioselective analysis of hydroxymebendazole (MBZOH), a chiral metabolite of mebendazole (MBZ), in human plasma by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The plasma samples were prepared by liquid-liquid extraction using diisopropyl ether: ethyl acetate (8 : 2, v/v). The HPLC chiral resolution of MBZOH was obtained using a chiral stationary phase based on tris-3,5-dimethyl-phenylcarbamate amylose derivative (Chiralpak AD column), under normal phase conditions using hexane/2-propanol/ethanol (81 : 14 : 5, v/v/v) as the mobile phase. CE resolution of MBZOH enantiomers was carried out in a fused-silica capillary (30 cm×75 μm ID) using 3.0% w/v sulfated β-cyclodextrin in 20 mmol/L Tris buffer, pH 7.0, as running buffer. Both methods were validated, showing mean recoveries of 70% and linearity in the 25–2500 ng/mL and 62.5–2500 ng/mL range for the HPLC and CE methods, respectively. The coefficient of variations and % relative error for both within-day and between-day assays were lower than 15% for both methods.