By combining HPLC-DAD-QTOF-MS and HPLC-SPE-NMR, the in vitro metabolism of vitetrifolin D, a pharmacologically active key molecule from Vitex agnus-castus in liver cell fractions, was investigated. Twenty-seven phase I and phase II metabolites were tentatively identified from the culture broth by HPLC-DAD-QTOF-MS. The subsequent HPLC-SPE-NMR analysis allowed for the unequivocal structural characterization of nine phase I metabolites. Since the preparative isolation of the metabolites was avoided, the substance input was much lower than in conventional strategies. The study did prove that the use of hyphenated instrumental analysis methodologies allows for the successful performance of in vitro metabolism studies, even if the availability of substances is very limited.