A high performance thin layer chromatography (HPTLC) method was developed and validated for determination of two anti-asthmatic drugs, salmeterol xinafoate and fluticasone propionate in co-formulations. Study was performed on pre-coated silica gel HPTLC plates using n-hexane:ethyl acetate:acetic acid (5:10:0.2) as a mobile phase. A TLC scanner set at 250 nm was used for direct evaluation of the chromatograms in reflectance/absorbance mode. Method was validated according to ICH guidelines. Determination coefficients of calibration curves were found 0.9977 and 0.9936 in the ranges 100–1000 and 200–2000 ng band1 for salmeterol and fluticasone, respectively. Method had an accuracy of 99.5% for salmeterol and 102.01% for fluticasone. Method had the potential to determine these drugs simultaneously from dosage forms without any interference.