The simultaneous determination of triamterene (TRM) and losartan (LOS) in their pure forms and in spiked human plasma was successfully achieved using a novel, simple, sensitive, and selective high-performance thin-layer chromatography (HPTLC) method with reflectance/absorbance mode for detection. This method has been validated and established according to International Council for Harmonization (ICH) criteria. Excellent separation of the coadministered drugs was accomplished using precoated silica gel 60 F254 as the stationary phase and a mobile phase consisting of toluene‒ethyl acetate‒methanol‒ acetone‒ammonia (6:1.5:1.5:0.9:0.1, V/V). This mobile phase mixture produced compact bands with good separation, yielding RF values of 0.19 for LOS and 0.35 for TRM. The mixture of TRM and LOS was quantified in a single run at 273 nm over concentration ranges of 90‒900 ng/band for LOS and 60‒600 ng/band for TRM. The limits of quantification were 72.33 ng/band for LOS and 43.76 ng/band for TRM, while the limits of detection were 23.87 ng/band and 14.44 ng/band for LOS and TRM, respectively. This validated method proved effective for the simultaneous determination of TRM and LOS in pure forms and could be successfully applied to spiked human plasma samples.