The anti-inflammatory drug ibuprofen (Ibu) is metabolized in the human liver to a number of metabolites including 1-hydroxyibuprofen (1-OH-Ibu), 2-OH-Ibu, and 3-OH-Ibu, respectively. The only human CYP known to produce relevant amounts of 3-OH-Ibu is CYP2C9 and as genetic polymorphisms of CYP2C9 influence the metabolization of numerous drugs, the availability of reference standards for CYP2C9-specific metabolites is of considerable interest. The aim of this study was to develop a biological production process for 3-OH-Ibu and to affirm its NMR characteristics. The recombinant fission yeast strain CAD68 coexpressing human CYP2C9 and CPR was used for the whole-cell biotransformation of Ibu to 3-OH-Ibu in 1L batch-scale for 75h. The average space-time yield for the bioproduction of 3-OH-Ibu (125±34μmol/Ld) considerably exceeded that of 2-OH-Ibu (44±10μmol/Ld). Accordingly, average biotransformation activities normalized to dry biomass weight were 5.0±0.8μmol/gd (3-OH-Ibu) and 1.9±0.7μmol/gd (2-OH-Ibu). The metabolite was prepurified on preparative TLC-plates, isolated by HPLC fractionation, and characterized by LC-MS and NMR. As expected, differential fragmentation patterns of 2-OH-Ibu and 3-OH-Ibu were detected in ESI-LC-MS analysis. 44mg of 3-OH-Ibu was efficiently purified from four 1L batch cultures and its structure was clearly confirmed by one- and two-dimensional NMR.