In order to determine the levels of metoclopramide hydrochloride (MTC), itopride hydrochloride (ITP), mosapride citrate (MR), and trimebutine maleate (TRI) in their pharmaceutical formulations and spiked human plasma, a new straightforward, accurate, highly sensitive, non-extractable, economical, and quick spectrofluorimetric method was developed. The technique was based on the reaction between the drug's tertiary amino group and eosin Y (EY), which causes quantitative fluorescence quenching of EY, which was detected at 542 nm after excitation at 520 nm. For MTC, ITP, and MS, the quenching in fluorescence intensity was directly proportional to the drug concentrations, while for TRI, it was between 100 and 1000 ng ml-1. And with a quantitation limit of 24, 40, 4, and 25 ng ml-1 for MTC, ITP, MR, and TRI, as well as detection limits of 8 ng ml-1, 13.5 ng ml-1, 12.1 ng ml-1, and 8.77 ng ml-1. According to ICH requirements, the proposed analytical method was validated, and the outcomes were satisfactory. The researched medicines' presence in pharmaceutical formulations and spiked human plasma has been successfully determined using the suggested analytical method, which also provides an excellent percent recovery. The outcomes are quite good. agreement with the reported method with respect to precision and accuracy