A novel, environmentally friendly, and sensitive HPTLC method has been developed and validated for simultaneous quantification of duloxetine (DLX) and tadalafil (TDL) in their pure state, laboratory-prepared mixtures, and spiked human plasma. This method is particularly important for patients dealing with depression and sexual issues, as it allows for the measurement of these co-administered antidepressant and sexual stimulant drugs in biological fluids. The separation process employed a stationary phase of pre-coated silica gel 60 F254 and a mobile phase consisting of ethyl acetate, acetonitrile, and 33% ammonia (8:1:1, v/v). The optimized mobile phase resulted in well-defined bands for DLX and TDL, with Rf values of 0.3 and 0.8, respectively with dual-wavelength detection at 232 nm for DLX and 222 nm for TDL. Polynomial regression analysis revealed exceptional linearity for both drugs, with correlation coefficients of 0.9999 over concentration ranges of 10–900 ng/band for DLX and 10-1200 ng/band for TDL. The quantitation limits were 8.2 ng/band for DLX and 8.6 ng/band for TDL, while the detection limits (LOD) were 2.7 ng/band for DLX and 2.8 ng/band for TDL. The validation of this method followed the guidelines set by the International Council for Harmonization (ICH). Additionally, the suggested method’s greenness was assessed by means of four up-to-date ecological tools, namely the Eco-Scale, the National Environmental Method Index (NEMI), the Green Analytical Procedure Index (GAPI), and the Analytical Greenness metric approach (AGREE). The proposed method was also assessed using the Blue Applicability Grade Index (BAGI), a recently developed metric for assessing the practicality (blueness) of procedures.