In this study, a flow cytometry (FC) protocol was implemented to measure poly(3-hydroxybutyrate) (PHB) content in a halophilic bacterium, to have a faster and easier control of the process. The halophilic bacterium Halomonas boliviensis was stained with BODIPY 493/503 and analyzed using FC. Bacterial polymer accumulation induced by two different nutrient limitations during the operation of a 2 L bioreactor was studied using traditional gas chromatography (GC) analysis and FC. The application of this rapid and straightforward method is useful to obtain complex and precise information about PHB accumulation that could be used for the monitoring, control and optimization of the production of PHB. A clear correlation between the PHB concentration determined by GC and the fluorescence signal obtained from stained bacteria by using FC was observed. Additionally, the heterogeneity of bacterial population as a function of PHB content was measured. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:276–284, 2017.