Background: Tamoxifen is a prodrug that will undergo metabolism by the CYP2D6 enzyme to become a more active metabolite, including endoxifen, N-desmethyltamoxifen, and 4-hydroxitamoksifen. Therefore, the effectiveness of therapy is determined by its metabolites. Volumetric Absorptive Microsampling is one of the biosampling methods that is comfortable for patients and able to analyze the concentration of metabolites in the blood.  Objective: This study aims to obtain an optimum and validated analysis method for tamoxifen and its metabolites in Volumetric Absorptive Microsampling using Liquid Chromatography - Tandem Mass Spectrometry. Materials and Methods: The UPLC-MS/MS method was developed and validated with propranolol as the internal standard. Optimization was done by evaluating several parameters that affect the efficiency of VAMS preparation and analysis of Tamoxifen and its metabolites. Results: Sample preparation was done by solvent extraction method and sonication-assisted extraction using methanol as extraction solvent. Separation was carried out by reverse phase chromatography using the Acquity UPLC BEH C₁₈ column (2.1 x 100 mm; 1.7 mm), with a flow rate of 0.2 mL/minute, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 minutes. Quantitative analysis of analytes was performed using triple quadrupole mass spectrometry with electrospray ionization (ESI) positive ion mode. The multiple reaction monitoring (MRM) value is set at m/z 358,22 > 58,09 for N-desmethyltamoxifen, m/z 372.2 > 72.27 for tamoxifen, m/z 388.29 > 72.19 for 4-hydroxitamoxifen, m/z 374.29 > 58.2 for endoxifen, and m/z 260.2 > 116.2 for propranolol hydrochloride. The lower quantification limit value (LLOQ) is 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 3.00 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Conclusion: This method proved to be valid, sensitive, and selective for the analysis of tamoxifen and its metabolites according to the US FDA’s Bioanalytical Method Validation Guidance.

Key words: 4-hydroxitamoxifen, endoxifen, UHPLC-MSMS, N-desmethyltamoxifen, propranolol, Tamoxifen, Volumetric Absorptive Microsampling.