This study aimed to develop LC-MS/MS method for PF-543, a selective SphK1 inhibitor and a promising anti-cancer agent for hepatocellular carcinoma, and to investigate the pharmacokinetic (PK) properties of PF-543 in mice. The LC-MS/MS method was developed and validated according to the guidance on bioanalytical method validation. The validated method was used to investigate the PKs of PF-543 in mice following its administration to mice intravenously and per oral route at doses of 5 mg/kg and 30 mg/kg, respectively. PF-543 was detected in ESI+ mode using an MRM transition of m/z 466.2 → 223.1. A calibration standard curve was linear within the concentration range of 0.1-100 ng/mL (r²=0.993) determined from linear regression analysis using a weighting of 1/x². The intra- and inter-day precision and accuracy met the acceptable criteria. PF-543 showed consistent recovery and minimal matrix effects and remained stable under short-term, freeze-thaw, and post-treatment conditions. Following PO administration, PF-543 was rapidly absorbed into plasma (mean T_max of 0.73 h) but was eliminated rapidly, resulting in detectable levels in the plasma for 8 h. Oral BA of PF-543 was calculated as 22.9%. Simple and sensitive bioanalytical method for PF-543 was validated and applied to the PKs study, supporting future PKs/pharmacodynamic analyses and safety assessments to better understand the therapeutic potential of PF-543 in tumor-bearing mice.