Overexpression of protein arginine methyltransferase 5 (PRMT5) is pivotal in MYC-driven primary medulloblastoma tumors, suggesting PRMT5 as a potential therapeutic target. JNJ, a potent PRMT5 inhibitor currently in clinical trials, notably for non-Hodgkin lymphoma and lung cancer, was evaluated in this study. We report a validated LC–MS/MS bioanalytical method for quantifying JNJ in plasma and tissue matrices. The method demonstrated acceptable sensitivity, selectivity, and robustness in accordance with regulatory guidelines. The assay was linear over the range 0.2–500 ng mL⁻¹ (r² = 0.99), with plasma recovery exceeding 84% using only 100 µL of sample. Precision (%RSD < 15%) and accuracy (~91–108%) were within acceptable limits. JNJ showed >94% plasma protein binding and moderate Caco-2 permeability (3.4 ± 0.4 × 10⁻⁶ cm s⁻¹). Hepatic intrinsic clearance was higher in mouse liver microsomes than in human (41 ± 19 vs. 7 ± 0.6 mL min⁻¹ kg⁻¹). Following oral dosing in mice (10 mg kg⁻¹), T_max was 30 min with a C_max of 2781 ± 1033 ng mL⁻¹. Oral bioavailability was low (15%). The validated method was successfully applied to in vitro and in vivo studies and will guide dosing in animal models of medulloblastoma.