This study introduces a novel and eco-friendly method for the spectrofluorimetric determination of two important anti-GERD drugs, esomeprazole and lansoprazole. The study depends on synthesis of silver nanoparticles from Anisosciadium seeds extract as a reducing agent, for the first time. The synthesized silver nanoparticles were characterized by applying various microscopic and spectroscopic methods, including Fourier Transform Infrared, High Resolution Transmission Electron Microscopy, Energy-Dispersive X-Ray Analysis, Dynamic Light Scattering technique, Ultraviolet–Visible spectrophotometry, and spectrofluorimetry. The produced silver nanoparticles demonstrated an emission band at 311 nm if excited at 222 nm, and showed powerful blue fluorescence when exposed to UV light. The obtained silver nanoparticles were employed as fluorescent nanoprobes for the determination of esomeprazole and lansoprazole spectrofluorimetrically, depending on their quenching of the native fluorescence of silver nanoparticles quantitatively. This study is the first spectrofluorimetric strategy for the determination of the investigated medications with the use of silver nanoparticles without the requirement for any pre-derivatization stages or large volumes of organic solvents. As investigated medications don’t display native fluorescent characteristics; the importance of the presented method increases. The suggested study demonstrated excellent linearity over the ranges of 1.0–10.0 μg/mL and 0.1–1.0 μg/mL with detection limits of 0.17 μg/mL and 0.015 μg/mL for esomeprazole and lansoprazole, respectively. The presented study was employed for the determination of esomeprazole and lansoprazole in human plasma samples and dosage forms with low % relative standard deviation and high % recoveries values. Greenness profile of the suggested method was assessed utilizing two various metrics, including Green Analytical Procedure Index (GAPI) and Analytical GREENNESS metric approach (AGREE), verifying lower environmental impact of the method and excellent eco-friendliness. The proposed strategy was validated in accordance with International Council for Harmonization(ICH) Q2 (R2) guidelines.