Leniolisib, a selective phosphoinositide 3-kinase δ (PI3Kδ) inhibitor, was recently approved for the treatment of activated PI3Kδ syndrome (APDS). A robust bioanalytical method is essential for pharmacokinetic (PK) studies and therapeutic drug monitoring. This study aimed to develop and validate a sensitive, selective, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying leniolisib in rat plasma. The method involved protein precipitation followed by LC-MS/MS analysis using gefitinib as an internal standard. Chromatographic separation was achieved on an Inertsil ODS column (150 mm × 4.6 mm, 3.5 µm) with a mobile phase of methanol and water (50:50, 0.1% formic acid). The method was validated per FDA guidelines for selectivity, linearity, precision, accuracy, recovery, matrix effects, and stability. The assay exhibited linearity over 10–80 ng/mL (R² = 0.9998). Intra- and inter-day precision (%CV) was <1.7%, and accuracy ranged from 94.98% to 98.03%. Recovery was consistent (~97%), with no significant matrix effects. Stability studies confirmed leniolisib's robustness under various storage conditions. Pharmacokinetic analysis in rats revealed a Tmax of 2 h, Cmax of 39.366 ng/mL, and t1/2 of 8 h. The validated LC-MS/MS method is precise, accurate, and suitable for pharmacokinetic studies of leniolisib in preclinical and clinical research.Pharmacokinetics studies in rat of leniolisib demonstrated that AUC0-t value is 318 ng-hr/ml while Cmax value is 39.366 ng/ml. The successful validation of bioanalytical method demonstrated that the PK parameters could be extrapolated from rats to human for administration of leniolisib.