Aims: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder. Methods and Results: The BactiFlow(TM) flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0.1% peptone were treated with 0.8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55 degrees C for 2-3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results. Conclusions: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer. Significance and Impact of the Study: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product.