A rapid, reliable and sensitive UV-spectrophotometric method has been developed for the determination of the Sulphamethoxazole and its in- vitro protein binding studies. The developed method has been validated statistically as per USP. The sulphamethoxazole shows λ max at 254.2 nm and the linearity plot yielded a correlation coefficient (R2) of 0.9999 over the Beers range of 1–5µg/ml. The regression equation was found to be Y=0.0676x + 0.0011, The molar absorptivity (mol/lit) was found to be 1.72X104 with Sandells sensitivity ( µg/cm2/0.001 ) of 0.01468, % recovery 100.42, %RSD 0.0041 for precision, LOD & LOQ were found to be 0.283 and 0.857 respectively. The developed method was employed in in- vitro protein binding studies, the drug shows 50-60% binding and it was good agreement with reported pharmacokinetic data and drug release kinetics.