Impurity profiling of active pharmaceutical ingredients is a crucial step in assessing their quality. In this study, a green stability indicating method for simultaneous determination of latanoprost (LTN) and timolol (TIM) was validated and assessed for greenness. TIM & LTN are two anti-glaucoma drugs manufactured as combination therapy. Their separation was done in presence of their pharmacopeial impurities including LTN C3-epimer using chiral mobile phase additives. Stress conditions were applied to affirm the stability indicating power of the LC-separation. Gradient elution was employed using mixture of aqueous phase (containing 11.35 g β-cyclodextrin and 1.5 g sodium octane sulfonate/L) and the green organic modifier ethanol. Separation was done on monolithic RP-C18 column and 210 nm UV-detection. Elution time was <18 min with retention times 4.9 and 16.1 min for TIM and LTN, respectively. The proposed method was validated for linearity, accuracy and precision and was found to be acceptable over the range of 50–750 and 1–50 μg/mL for TIM and LTN, respectively. The method was then applied successfully to determine TIM and LTN in marketed pharmaceutical ophthalmic solution. Since claiming greenness is not enough, two assessment tools (Analytical eco-scale and Green Analytical Procedure Index; GAPI) were utilized to compare our proposed method to other reported methods.