Doxorubicin is a broad spectrum anthracycline antibiotic glicoside which has antineoplastic activity. This chemotherapy agent is the most administrated agent to medicate solid tumour in adults including lungs, ovarium, and breast cancer also malignant lymphoma. Long term use of this chemotherapy agent is limited because development of progressive dose-dependent cardiomyopathy which develops irreversibly towards congestive heart failure. The cardiotoxic effect of doxorubicin is highly determined by accumulation of its main metabolite, doxorubicinol. The purpose of this study was to obtain a validated analysis method of doxorubicin and doxorubicinol simultaneously in plasma with hexamethylphosphoramide as the internal standard using liquid chromatography-tandem mass spectrometry, therefore optimization and full validation are conducted in this research. Sample preparation was performed by protein precipitation using methanol. The separation was performed using UPLC C-18 BEH (2.1 x 100 mm), 1.7 μm column, 45⁰C temperature column. The mobile phase consisted of 0.1% acetate acid (eluent A) and acetonitrile (eluent B)  at 0.15 ml/min with gradient elution. The detection of the mass was performed on Waters Xevo TQD using ESI positive (+) type MRM for doxorubicin, doxorubicinol, and hexamethylphosphoramide with m/z values: 544.22>361.05; 546.22>363.05; and 180.03>135.16, respectively.  This method is linear in the range concentration of 1-100 ng/mL for doxorubicin with r value = 0.9963 ; 0.5 -50 ng/mL for doxorubicinol with r value = 0.9977 . %diff and %CV of the assay were less than 20% for LLOQ and less than 15% for other concentration. LLOQ for doxorubicin and doxorubicinol 1.0 ng/mL and 0.5 ng/mL respectively. This method has successfully fulfilled validation requirement refers to EMEA Guidelines (2011) and FDA guidelines (2018).