The pharmacokinetics of LXT-101 sustained-release suspension, an anti-prostate cancer polypeptide, were studied on beagle dogs using a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Samples were prepared by the protein precipitation, evaporation and reconstitution. Chromatographic separation was performed using a Hypersil GOLD C18 column (50 mm × 2.1 mm, I.D. 5 μm). The mobile phase consisted of acetonitrile, water and formic acid (20:80:0.1, v/v/v) at a flow rate of 0.3 mL/min. The acquisition was carried out in selected reaction monitoring (SRM). The method was validated in terms of selectivity, linearity, precision and accuracy, extraction recovery and matrix effect, and stability. It showed good linearity over the range of 2-600 ng/mL (R2 = 0.9977). The intra- and inter-batch accuracy were within 93.36-93.94% and 95.61-99.27%, while the intra- and inter-batch precision were in the range of 3.23-14.26% and 5.03-11.10%, respectively. The extraction recovery and matrix effect data for LXT-101 in beagle dog plasma ranged from 75.90-126.40% and 83.13-95.50%, respectively. The stability results proved that the storage conditions, disposal, intermittent analysis and analysis techniques were valid and reliable for LXT-101 in beagle dog plasma. In the single-dose groups (20 mg/kg and 40 mg/kg), the values of AUC0-t (588.09 ± 137.79 ng/mL·d vs. 1203.62 ± 877.42 ng/mL·d) and AUC0-∞ (592.89 ± 134.21 ng/mL·d vs. 1209.97 ± 873.78 ng/mL·d) were observed increasing proportionately with the increasing dose of LXT-101 sustained-release suspension. The results in the repeated-dose group suggested the possibility of accumulation in beagle dogs.