[(E)-3-(2-chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1,2,3,6-tetrahydropyridine-4-yl)phenyl)-2-propylene-1-one], L41, was found to be a potent cyclin-dependent kinase 1 inhibitor. To study its pharmacokinetic characteristics, a simple, specific, sensitive and reproducible HPLC method was developed and validated to quantitatively determine L41 in rat plasma. L41 and amlodipine besylate (internal standard, IS) were extracted from rat plasma by a simple liquid–liquid extraction process with ethyl acetate. Chromatographic separation was performed on an Agela C₁₈ column and an isocratic mobile phase [methanol–water (containing 50 mM ammonium acetate), 77 : 23, v/v, pH 6.5] at a flow rate of 1 mL min⁻¹ with a total run time of 10 min. The UV absorbance at 343 nm was recorded. L41 and IS eluted at 5.1 and 8.9 min, respectively. The calibration plot was linear over the concentration ranging of 15–1200 ng mL⁻¹ (r > 0.998). The intra- and inter-day precisions of analysis were <12% and accuracy ranged from 93.5 to 106.3%. The validated HPLC method was successfully applied to the pharmacokinetic study of L41 in rats after a single intravenous and three oral administrations.