A bioanalytical method for simultaneous estimation of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human K3 EDTA plasma is described. An API 3000 mass spectrometer was employed in this method where olmesartan d4 (OLMD4) and hydroflumethiazide (HFTZ) served as the internal standard. Sample preparation involved solid phase extraction (SPE), a polymer based, hydrophilic-lipophilic balanced cartridges, and chromatographic resolution achieved on X Terra RP18, (4.6 × 150 mm, 5 µm) column using a mobile phase of 2 mM ammonium formate buffer, (pH 3.50 ± 0.10 with formic acid)/acetonitrile (30:70, v/v). Negative mass transitions (m/z) of OLM, HCTZ, OLMD4, and HFTZ were detected in multiple reactions monitoring (MRM) mode at 445.6 → 148.4, 295.9 → 268.9, 449.4 → 148.4, and 329.9 → 238.3, respectively. The linearity was checked over a concentration range of 0.11 × 101 to 1.06 × 103 ng/mL for OLM and 0.10 × 101 to 0.32 × 103 ng/mL for HCTZ. Intra- and inter-run precision of OLM and HCTZ assay at four concentration levels were below 8.1% and 8.0%, and accuracy was within ±3.6% and 9.0%, respectively. Recoveries for OLM, HCTZ, and internal standards OLMD4 and HFTZ were 70.4, 63.5%, and 97.1, 75.2%, respectively. The pre-validation exercises for OLM/HCTZ in human plasma were done using special conditions such as ice cold water bath under low light.