The native fluorescence of betaxolol (a potent drug for hypertension and glaucoma) has been studied under different experimental conditions The highest fluorescence intensity vas obtained in methanol at 305 nm using 25 nm for excitation Surfactants and sensitizers had either negative or no effect on its fluorescence intensity .The fluorescence intensity-concentration plot was rectilinear over the range 0.1-1.0 µg mL^-1 with lower detection limit of 0.025 µg mL^-1 (8.1 x 10^-8 M) Interference likely to be introduced from co-formulated drugs (such as benzalkonium chloride) or co-administered drugs (such as hydrochlorothiazide), was studied The method was successfully applied to the determination of the drug in its tablets and ophthalmic solution The mean % recoveries were in agreement with those provided by the official methods. The method was further extended to the in-vitro determination of the drug in spiked human plasma using solid phase extraction, utilizing a C₁₈ Bond-Flute column. The mean % recovery (n=5) was 101.38 ±2.32.