This study presents the development and validation of a novel high-performance liquid chromatography method employing fluorescence detection for the rapid determination of indoleacetic acid (IAA) in bacterial cell cultures. IAA, a pivotal plant growth hormone, influences various physiological processes and is often employed as a marker to screen the growth-promoting properties of rhizosphere and endophytic bacteria. Current methods for IAA determination face challenges such as lengthy and costly sample preparations, complex matrix interferences, and the need for expensive equipment. This method utilizes simple sample preparation and rapid analysis procedures, mitigating previous limitations. The method’s performance was assessed using two bacterial strains, Bacillus subtilis 26D and Bacillus amyloliquefaciens IMV A4. The method was also validated according to ICH guidelines, confirming specificity, linearity, accuracy, and repeatability. The developed method demonstrated significant improvements in speed and environmental impact, utilizing minimal solvent volumes and offering quick turnaround times. This method is a valuable tool for the rapid screening of bacterial strains for IAA production, facilitating the optimization of growth conditions in agricultural biotechnology applications.