It was the first time that a spectrofluorimetric approach for the simultaneous analysis of remdesivir and levodropropizine had been achieved. This study aims to propose an accurate and sensitive second-derivative synchronous spectrofluorimetric approach for measurement of remdesivir and levodropropizine in different matrices simultaneously without the need for prior separation. The proposed approach measured the synchronous fluorescence intensity of pharmaceuticals under research at a constant wavelength difference (Δλ) = 130 nm. For the quantitative analysis of remdesivir and levodropropizine, the peak amplitudes of the second derivative were measured at 390 and 399 nm, respectively. The procedure was completely validated and demonstrated outstanding linearity in the concentration ranges of 5–150 ng mL− 1 and 10–600 ng mL− 1 for remdesivir and levodropropizine, respectively. The new method was used to quantitatively analyze both drugs in their pharmaceutical dosage form, synthetically formulated mixture and spiked human plasma. A statistical comparison of the results with other published analytical techniques revealed no significant difference. The validation of the procedure was successfully completed in compliance with ICH guidelines. In terms of greenness, EcoScale and GAPI greenness tools were used to evaluate the analytical methodology.