Six new, simple, rapid, sensitive, accurate, precise and economical spectrophotometric methods were developed and validated for simultaneous determination of Lamivudine (LAM) and Tenofovir (TEN) in bulk and pharmaceutical dosage forms. Method A is Simultaneous equation method, where LAM and TEN were determined at 270.8 nm and 260.6 nm. Method B is first derivative zero crossing point method (FDZ) where LAM and TEN were determined at 260 nm and 249 nm, respectively. Method C is second derivative dual wavelength (DWL) where ∆A at 255 nm and 269 nm for LAM≠0 and ∆A at 281 nm and 267 nm for TEN ≠0. Method D is first derivative of ratio spectra derivative method, where peak amplitudes at 223.80 nm for LAM and 245.60 nm for TEN were used. Method E is ratio difference spectroscopic method where ∆A at 255.2 nm and 210.6 nm (TEN 3 µg/ml is used as the divisor) of the zero order divisor spectra were measured for the determination of LAM. Whereas ∆A at 255.4 nm and 283.8 nm (LAM 9 µg/ml is used as the divisor) of the zero order divisor spectra were measured for the determination of TEN. Method F is amplitude factor method where LAM is determined at 266nm and TEN was determined at 393 nm. The two compounds were simultaneously determined in the concentration ranges of 3-15 µg/ml for both LAM and TEN. The methods were validated according to the ICH guidelines.