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The present research conveys an innovative, affordable, simple, and environmentally sustainable multivariate spectrophotometric method for the contemporaneous determination of timolol, brimonidine, and brinzolamide in their co-formulated pharmaceutical formulations and artificial aqueous humor. The studied drugs are widely prescribed either alone or in combination for the effective treatment of glaucoma to prevent the development of progressive glaucoma and visual neuropathy. The five multivariate models that were employed in the analysis were CLS, PCR, PLS, GA-PLS, and ANN. A five-level, three-factor experimental design was implemented to create an assortment of mixtures in the range between 4.0-16.0 µg/mL for timolol, 3.0-5.0 µg/mL for brimonidine, and 4.0-36.0 µg/mL for brinzolamide. The validity of the proposed methodology was evaluated through a set of diagnostic tools, including percentage recoveries, standard deviation, correlation coefficient, the root mean square error of prediction, and the root mean square error of calibration. For timolol, the study revealed that the root mean square errors of prediction for the CLS, PCR, PLS, GA-PLS, and ANN models were, respectively 0.086, 0.072, 0.066, 0.051, and 0.161. The equivalent brimonidine readings were 0.017, 0.031, 0.018, 0.016, and 0.063, respectively. While for brinzolamide, the corresponding findings for the five models were 0.188, 0.191, 0.0106, 0.123, and 0.307, respectively. These outcomes reveal how precise and accurate the generated models were. The reasonable sensitivity of the adopted method enables the analysis of studied medications in artificial aqueous humor. The environmental sustainability of the proposed methodology was assessed through diverse metrics including analytical eco-scale, AGREE, and GAPI.