AIM: To determine the concentration of ARV-471 (a representative PROTAC drug) in mice plasma by establishing an HPLC-MS/MS method and to apply this method in pharmacokinetic studies in mice. METHODS: Verapamil was taken as the internal standard, the mice plasma samples were extracted by methanol. After centrifugation, the supernatant was analyzed by liquid chromatography-mass spectroscopy (LC-MS/MS). Chromatographic column: ACQUITY UPLC HSS T3 (1.8 μm, 2.1 mm×50 mm); The mobile phase consists of acetonitrile (containing 0.1% formic acid) and 2 mmol/L ammonium formate aqueous solution (containing 0.1% formic acid) for gradient elution; flow-rate of 0.6 mL/min; injection volume: 2 μL. The electrospray ionization (ESI) is employed, operating in positive ion scanning mode with multiple reaction monitoring (MRM) detection. The specificity, standard curve and quantification limit, precision and accuracy, recovery rate and matrix effect, stability and dilution reliability of this method were examined. Furthermore, the plasma concentration and pharmacokinetic parameters of ARV-471 in mice after intravenous injection of 5 mg/kg and oral gavage of 30 mg/kg were determined and calculated. RESULTS： The results demonstrate that ARV-471 exhibits a good linear relationship within the concentration range of 2.0 to 2 000.0 ng/mL. The intra- and inter- accuracy were between 80.0% and 120.0%, with the intra- and inter- precision less than 15%. The results of methodological study of specificity, matrix effect, stability conformed to the requirements of the guideline. Pharmacokinetic parameters reveal that after oral administration of 30 mg/kg ARV-471 to male and female mice, Cmax were (2 947.19 ± 454.77) and (2 682.02 ± 342.23) ng·mL-1; AUC0-t were (23 357.37 ± 3 488.00) and (20 161.23 ± 1 871.32) ng·h·mL-1; T1/2 were (3.11 ± 0.18) and (2.93 ± 0.62) h. Tmax were (1.83 ± 0.41) and (2.00 ± 0.00) h for male and female mice, respectively .After intravenous administration of 5 mg/kg ARV-471 to male and female mice, the AUC0-t values were found to be (18 219.07 ± 2 059.41) and (17 238.01 ± 2 380.55) ng·h·mL-1; T1/2 values were (2.76 ± 0.23) and (2.73 ± 0.20) h; The absolute bio-availability of ARV-471 were determined to be (19.49 ± 1.81) % and (21.37 ± 3.19) % for male and female mice, respectively. CONCLUSION： This study establishes a rapid and effective method for the pharmacokinetic research of ARV-471, laying the foundation for the pharmacokinetic studies of PROTAC drugs.