Cytarabine (Cyt) (also known as cytosine arabinoside (ara-C)) used in the treatment of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL). CYT applied in high doses for treatment can cause renal failure. Monitoring excreted urine drug levels can help kidney failure. For this reason, a method was developed and validated by HPLC-MSMS for urine CYT analysis, which is not included in the literature. In this study, a liquid chromatography (HPLC) with triple quadrupole Mass Spectrometric (MS/MS) method developed for the determination of Cyt from urine for toxicokinetic evaluation. Positive MRM mode selected for the quantification of Cyt. The product and major fragment ion for Cyt 244.0 > 112.0 m/z, for IS 198.0 > 152.0 m/z. The optimal MS parameters for Cyt and IS are as follows Fragmentor 80 V, 70 V, Collision energy, 6, 9 respectively. A novel simple, high-throughput and highly sensitive HPLC-MS/MS method was successfully developed and validated for the determination of Cyt from urine. The developed method has a simple one-step extraction method and a short run time (2.0 minutes) for analysis. The proposed method could be practical and reliable for excretion and toxicokinetic studies and as well as the Therapeutic Drug Monitoring study in humans without an invasive route for Cyt.