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The rapid and sensitive detection of microbial contaminants is critical for timely contamination investigations and monitoring of process control for in vitro protein production systems. A flow cytometric method was developed to monitor two types of cell culture media used in large scale protein production. The process used a DNA dye, thiazole orange, which binds to nucleic acids of viable and nonviable organisms. To ensure a representative sample was tested and to enhance the detection limit, a concentration step before staining and analysis included a double centrifugation. In addition, a washing step was included to eliminate background fluorescence caused by material in the media formulations. The staining and analysis of concentrated and washed samples takes approximately 0.5 h and provides objective results. The feasibility of the method was demonstrated by spiking sterile media with six bacterial species that represent the most commonly encountered bacterial contaminants. In addition to the bacterial spiking study, 164 lots of large scale production medium were tested by the flow cytometric method in parallel with conventional culture using Trypticase Soy Broth (TSB). In the bacterial spiking study, the concentration method increased the microbial titer by 2 logs. The detection limit of organisms within the medium was determined to be 1 CFU/ml. There was 100% correlation between the flow cytometric method and the standard aerobic plate count method using Trypticase Soy Agar. In the parallel study on 164 lots of large scale production media, the flow cytometric method was compared against culture in TSB. The sensitivity of the method was determined to be 100%, the specificity was 99.4%, the positive predictive value was 83%, and the negative predictive value was 100%. To reduce reporting of false positives, an initial positive result should be confirmed by an additional sample. The analytic sensitivity, specificity, and objectivity of this flow cytometric method indicate this method is valuable in the monitoring of production media for microbial contamination.