Background: The 6-mercaptopurine is an active ingredient used to treat certain types of leukemia. This drug is an immunosuppressive and antineoplastic agent that belongs to the thiopurine class. In Brazil, 6-MP is currently available only in the form of 50 mg tablets, sold as Purinethol® and manufactured by Glaxo Smith Kline. The lack of the liquid formulation’s production impedes treatment, assuming that one of its advantages go through its applicability in pediatric patients, which shows the highest incidence among others. Objective: The purpose of this work was to evaluate the development and application of a reverse phase high performance liquid chromatography (HPLC) method using an Agilent 1220 Infinity® G4294B chromatograph with photodiode array detector. Methods: HPLC assays were performed on an Eclipse plus® C18 column (4.6 x 150 mm, 3.5 µm particle size) using a gradient mode mixture of acetonitrile and aqueous acetic acid solution as a mobile phase, with a flow of 1 mL.min-1 and detection at 324 nm. The method was validated by determining its selectivity, linearity, precision, accuracy, and robustness. Results: Retention time for 6-mercaptopurine was 5.12 minutes. The detector’s response was linear at concentrations from 1.6 to 2.4 µg/mL. The results of the method’s accuracy evaluation of the accuracy showed with mean recovery of the amount of substance added to the samples of between 99.88 and 100.5%. For precision, repeatability and intermediate precision were evaluated. The repeatability showed standard deviation of 0.0737. The intermediate precision was assessed on three different. For the three days of the studies, the values of the standard deviations were less than 3%, showing repeatability and intermediate precision adequate for the analytical method in question. The limit of detection was determined as 3.6 ng/mL. The limit of quantification was determined as 12 ng/mL. The chromatographic method was robust under the proposed Conclusion: The proposed method can be applied to quality control of 6-MP oral suspension to ensure that the required content is delivered to pediatric oncology patients.