(E)-3-(2-Chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1,2,3,6-tetrahydropyridine-4-yl)phenyl)-2-propylene-1-one (L41) is a novel synthetic cyclin-dependent kinase 1 inhibitor. In this study, we proposed a strategy to systematically characterize associated phase I and phase II metabolites of L41 using rat plasma and urine samples. Biological samples were treated with an optimized sample preparation approach involving protein precipitation, liquid–liquid extraction and solid-phase extraction prior to LC–MS/MS analysis. Both an Agilent LC/MSD Trap VL and an AB SCIEX API 5000 triple quadrupole mass spectrometer were used for LC–MS/MS analysis. Based on the proposed strategy, 3 phase I and 4 phase II metabolites were detected by a highly sensitive MRM method. The major metabolites of L41 were found to be demethylation metabolites (M5, M6), C-4′,6′-di-demethylation metabolites followed by a C-2′ (OH) methylation metabolite (M7), and were shown to undergo subsequent glucuronidation processes. These results provide evidence for in vivo L41 metabolism, and the method may be applied to the further analysis of L41 and its metabolites in biological samples.