Green analytical technologies for the determination of a bioactive compound diosmin (DIOM) in the real samples of pharmaceutical formulations and biological fluids are scarce in literature. Therefore, the present investigation was carried out to develop a novel, rapid, simple, and economical green “reversed phase high-performance thin-layer chromatography (RP-HPTLC)” method for the determination of DIOM in commercial tablets and in-house developed spray-dried microparticles (MPs). The quantification of DIOM was conducted via “RP-18 silica gel 60 F254S HPTLC plates”. The binary combination of green solvents, i.e., ethanol:water (5.5:4.5 v/v) was proposed as a green mobile phase. The analysis of DIOM was conducted in absorbance/reflectance mode of densitometry at λmax = 348 nm. The densitograms of DIOM from the commercial tablets and in-house developed spray-dried MPs were verified by recording their single band at Rf = 0.80 ± 0.02 compared to standard DIOM. Green RP-HPTLC method was observed as linear in the range of 100−700 ng/band with R2 = 0.9995. The proposed method was found as “accurate, precise, robust, and sensitive” for the determination of DIOM in the real samples of commercial tablets and in-house developed spray-dried MPs. The % content of DIOM in the real samples of commercial tablets and in-house developed spray-dried MPs was obtained as 99.06 and 101.30%, respectively. The recorded results of this research suggested that the green RP-HPTLC method can be effectively used for the routine analysis of DIOM in pharmaceutical products.