This study presents a novel derivatization-free spectrofluorimetric method for the quantification of vonoprazan (VPZ) that is characterized by its high sensitivity, environmental friendliness, and cost-effectiveness. Notably, this method eliminates the requirement for pre-derivatization, extraction procedures, large amounts of organic solvents, and/or expensive instrumentation, which distinguishes it from previous approaches. The approach employed in this study relies on the formation of an association complex between VPZand the xanthene dye acid red 87 (AR87) under acidic conditions. The stability of the VPZ-AR87 complex was determined to be remarkably high, with a complex formation constant (Kf) value exceeding 2 x 106 M−1. The fluorimetric measurements rely on the quantitative and selective quenchingof the dye’s native fluorescence emission at 545 nm. The experimental parameters were optimized utilizing a quality-by-design 23 full factorial design. This strategy yielded a highly favorable linearity range of 10.0 to 400.0 ng/mL, with a correlation coefficient (r) of 0.9997. The presented methodology exhibited a high level of sensitivity, as evidenced by its ability to detect concentrations as low as 1.2 ng/mL. This sensitivity enabled its successful application for the estimation of VPZ in human plasma samples with high %recoveries (98.21–101.67) and low %RSD values (<1.52). Furthermore, content uniformity testing of low-dose VPZ tablets was performed according to United States Pharmacopeia guidelines. Greenness and whiteness metrics analysis showed a significant advantage of the proposed method over previously reported fluorimetric methods. Validation of the developed approach was carried out in accordance with ICHQ2 (R1) guidelines.