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It was the first time that a spectrofluorimetric approach for the simultaneous analysis of methotrexate (MET) and 6-mercaptopurine (6-MP) had been achieved. This study aims to propose an accurate and sensitive second derivative synchronous spectrofluorimetric approach for the measurement of MET and 6-MP 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 6-MP and MET, the second derivative's peak amplitudes were observed at 380 and 400 nm, respectively. The process was fully validated and showed exceptional linearity for either 6-MP or MET in the concentration range of 10-100 ng mL^-1. The new method was used to quantitatively analyze both drugs in a synthetically formulated mixture, dosage forms, 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. To assess the analytical methodology's greenness, Eco-Scale and GAPI greenness tools were employed.