6-Mercaptopurine is a chemotherapeutic agent belongs to antimetabolite class. 6-Mercaptopurine has to go through the metabolic pathway to form 6-methylmercaptopurine. This study aimed to obtain an optimum and validated method in analyzing 6-MP and 6-MMP simultaneously in Dried Blood Spot samples and evaluate the potential for future drug concentration monitoring in DBS samples. The quality control and calibration curves were made by spotting 40 µL blood at DBS paper and dried for 3 hours. DBS papers were cut with a diameter of 8 mm and extracted with acetonitrile-methanol (1:3) containing internal standard 5-fluorouracil. Separation was performed with Waters Acquity UPLC BEH C18 column 1.7 μm (2.1 x 100 mm) with a mobile phase consists of 0.1% formic acid in water – 0.1% formic acid in acetonitrile with gradient elution and flow rate 0.2 mL/minute. Mass detection was done using Waters Xevo TQD with positive electrospray ionization (ESI) for 6-MP and 6-MMP and negative ESI for 5-FU in Multiple Reaction Monitoring mode. Detection of 6-MP, 6-MMP, 5-FU respectively were 153.09> 119.09; 167.17> 126.03; 129.09> 42.05. This method is linear with the range 26-1000 ng/mL for 6-MP and 13 to 500 ng/mL for 6-MMP with consecutive r value is ≥ 0,998 and ≥ 0,999. %Diff value and coefficient of variation (CV) for accuracy and precision of intra-day and inter-day are not more than 15% and not more than 20% at LLOQ concentration. This method also fulfilled the requirements of selectivity, linearity, carry-over, and matrix effects refers to the EMEA Guidelines.