Two simple, sensitive and accurate methods were developed for the determination of some antimicrobial substances. The first method is based on the measurement of difference in absorbance (?A) of equimolar portions of gatifloxacin solution in 0.1MHCl and in 0.1MNaOH at 291 nm. BeerÂs law is obeyed over a concentration range of 1- 9 µg ml -1with mean recovery 99.03 ± 0.03. The second method is depended on the measurement of a native fluorescence of Ofloxacin upon excitation at 290 nm with emission bands havingmaxima at 488 nmand found to be proportional to the concentration range 0.2 -2 µg ml-1 for Ofloxacin. Regression analysis showed good regression coefficient. The proposed methods were successfully applied for the determination of studied drugs in bulk powder and pharmaceutical dosage forms with good accuracy and precision. The results were found to agree statistically with those obtained by the official methods. Furthermore, the methods were validated according to ICH regulations and also assessed by applying the standard addition technique.