In the present study, a specific and sensitive liquid chromatography-triple quadrupole mass spectrometry method was developed and validated for the determination of SP-141, a novel pyrido[b]indole anticancer agent. After a liquid-liquid extraction with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) mixture, the analyte was separated on a Kinetex C18 column (50×2.1mm, 2.6μm) with mobile phases comprising of water (0.1% formic acid, v/v) and acetonitrile (0.1% formic acid, v/v) at a flow rate of 0.4mL/min. The test compound (SP-141) and the internal standard (SP-157) were analyzed in the multiple reaction-monitoring mode using the mass transitions m/z 325.1 → 282.0. The method was linear in the concentration range of 0.648-162ng/mL with coefficients of determination (R(2)) of 0.999 in mouse plasma. The lower limit of quantification was 0.648ng/mL. The intra- and inter-day assay precisions (coefficient of variation, %CV) were less than 4.2% and accuracies (relative error, %RE) ranged from -6.1% to 2.1%. The extraction recoveries were between 97.1 and 103.1% and the relative matrix effect was minimal. In addition, SP-141 was found to be stable in the plasma after three freeze-thaw cycles, at 37°C and 4°C for 24h, and at -80°C for 4 weeks. It was also stable in the stock solution at room temperature for 24h and after preparation in the autosampler for 36h. The validated method was successfully applied to an initial pharmacokinetic study of SP-141 in CD-1 mice following intraperitoneal and intravenous administrations.