In this study, determination of terbinafine and itraconazole down to biological concentration level has been carried out. The determination is based on increasing the selectivity of the spectrofluorimetric technique by combining both derivative and synchronous spectrofluorometric approaches which permits successful estimation of terbinafine at 257 and itraconazole at 319 in presence of each other at  λ of 60 nm. ICH validation guidelines were followed to fully validate the method, and linearity was obtained for the two drugs over the range of 0.1-0.7 g/mL for terbinafine and 0.5-4.0 g/mL for itraconazole. Application of the method was successfully carried out in the commercial tablets with good agreement with the comparison spectrofluorometric methods. As the detection limits were down to 0.013 and 0.1 g/mL and quantitation limits were 0.04, 0.032 g/mL for terbinafine and itraconazole, respectively; the in-vitro determination of terbinafine and itraconazole in spiked plasma samples was applicable. The percentage recoveries in biological samples were 97.17 ± 4.54 and 98.75 ± 2.25 for Terbinafine and Itraconazole respectively. Water was utilized as the optimum diluting solvent in the proposed methodology which adds an eco-friendly merit.