The proposed study introduces a rapid, sensitive, and simple synchronous spectrofluorimetric technique for simultaneous quantification of relebactam, cilastatin, and imipenem in marketed pharmaceutical forms and biological fluids. Using synchronous fluorescence spectroscopy at Δ λ = 110 nm, cilastatin was detected at 360 nm. Fourier Self-Deconvolution was subsequently applied to the spectrum to estimate relebactam and imipenem at 430 nm and 470 nm, respectively after detection of cilastatin at 360 nm ensuring no cross-interference. The pH was adjusted to 8.0 using 2.0 mL of alkaline borate buffer. This approach allowed for the precise quantification of relebactam, cilastatin, and imipenem through ranges of 50–400 ng mL− 1, 20–500 ng mL− 1, and 50–500 ng mL− 1 respectively. The lower detection and quantitation limits were 9.9 and 29.7 ng mL− 1 for REL, 4.5 and 13.6 ng mL− 1 for CIL and 5.5 and 16.5 ng mL− 1 for IMP. The proposed method was successfully applied for the determination of studied drugs in their pharmaceutical formulations with a high degree of accuracy and without interference from common excipients. This approach allowed for the precise quantification of relebactam, cilastatin, and imipenem through ranges of 50–400 ng mL− 1, 20–500 ng mL− 1, and 50–500 ng mL− 1, respectively. The proposed method was rigorously validated according to ICH guidelines. Furthermore, the method’s environmental impact was assessed using Eco-scale and Green Analytical Procedure Index (GAPI) techniques.