Three spectrophotometric methods have been developed and validated for the simultaneous estimation of a binary mixture of brimonidine tartrate (BRM) and timolol maleate (TML) in the presence of benzalkonium chloride (BNZ), namely derivative spectrophotometry (Method 1) calculated by numerical differentiation, Ratio spectra derivative zero crossing calculated by numerical differentiation (Method 2), and mean centering of ratio spectra (Method 3). Method 1 was applied to determine BRM at 286 nm, which shows zero crossing point with TML and BNZ, and TML was determined at 310 nm, which shows zero crossing point with BRM and BNZ. Method 2 was used to determine BRM and TML at 221.87 and                       258.43 nm, respectively.  Method 3 was applied for the determination of BRM at 221.9 nm and TML at 286.9 nm. BRM exhibited good linearity over the concentration range of 1.50 - 9.50 μg/mL for the three methods. TML was determined in the concentration range of 5.00 – 7.60 μg/mL,  5 – 10 μg/mL and 5.00 – 9.20 μg/mL for Methods 1, 2 and 3, respectively. The proposed methods have the requisite accuracy, selectivity and precision and were successfully applied for the determination of BRM and TML in the presence of BNZ in their pure forms, laboratory prepared mixtures and combined dosage forms.  The results obtained for the analysis of both drugs in their pure forms by the proposed methods were statistically compared to those obtained by applying a reported HPLC method.