In this paper, an improved spectrofluorimetric method for the simultaneous and direct determination of ammonium and primary amine compounds is presented. The method is based on the derivatization of the analytes with o-phthaldialdehyde (OPA)/N-acetylcysteine (NAC) reagent using high throughput microplates, and OPA/NAC ratio has been optimized in order to suppress interference of ammonium on primary amine determination. Direct measurement of these two parameters is therefore possible with a global procedure time that does not exceed 10 min. Excellent limits of detection of 1.32 μM and 0.55 μM have been achieved for ammonium and primary amines, respectively. Reagent stability issues have also been addressed and formulation of reagents solution is described for improved reagents shelf life. The proposed protocol was finally applied and validated on real samples such as wine samples, compost extracts and wastewater.