<jats:title>Abstract</jats:title><jats:p>Hollow fiber liquid‐phase microextraction and CE were applied for the determination of albendazole sulfoxide (ASOX) enantiomers in liquid culture medium after a fungal biotransformation study. The analytes were extracted from 1 mL of liquid culture medium spiked with the internal standard (<jats:italic>rac</jats:italic>‐hydroxychloroquine) and buffered with 0.50 mol/L phosphate buffer, pH 10. The analytes were extracted into 1‐octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 0.05 mol/L <jats:italic>tris</jats:italic>(hydroxymethyl)aminomethane buffer, pH 9.3, containing 3.0% w/v sulfated‐β‐CD (S‐β‐CD) with a constant voltage of +15 kV and detection at 220 nm. The method was linear over the concentration range of 250–5000 ng/mL for each ASOX enantiomer. Within‐day and between‐day assay precision and accuracy for the analytes were studied at three concentration levels and the values of RSD% and relative error % were lower than 15%. The developed method was applied for the determination of ASOX after a biotransformation study employing the endophytic fungus <jats:italic>Penicillium crustosum</jats:italic> (VR4). This study showed that the endophytic fungus was able to metabolize the albendazole to ASOX enantioselectively. In addition, it was demonstrated that hollow fiber liquid‐phase microextraction coupled to CE can be an excellent and environmentally friendly technique for the analysis of samples obtained in biotransformation studies.