Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and low-solvent-consumption technique. However, almost every mass in the low mass-to-charge-ratio region of the mass spectrum appears as strongly fluctuating matrix background signals. Thus, it is difficult to identify small molecules using this technique. In this study, we used methanol to methylate valsartan, an angiotensin II receptor blocker that is commonly used to treat high blood pressure and heart failure. The methylation derivatization of valsartan enhanced the detection sensitivity and transformed the detection m/z ratio. The liquid-phase microextraction of valsartan in human plasma (20 μL) was achieved by acidifying valsartan with HCl aqueous solution and extracting it with toluene. An acetyl chloride/anhydrous methanol mixture was added for methylation derivatization, which was completed within 30 min at 30 °C. Finally, the residue was re-dissolved in irbesartan methanolic solution, which together with the matrix 2-mercaptobenzothiazole was spotted on an AnchorChip target plate for MALDI-TOF MS analysis. Liquid-phase microextraction was performed and the methylation-derivatization parameters were investigated. The valsartan calibration range was 0.2-10 μg mL-1 with good linearity in human plasma. In the within- and between-run analyses, the relative standard deviation and relative error were both <11.32%. This method was successfully applied to determine the valsartan concentration in the plasma of 10 patients with hypertension.