The correlation between alkaline phosphatase (ALP) inactivation and bacterial pathogen inactivation in raw milk was studied using a newly Association of Official Analytical Chemists-approved fluorometric assay (AOAC 991.24). Fresh, raw milk was inoculated with Listeria monocytogenes Scott A and Salmonella senftenberg 775W at levels of approximately 1 × 104 and 1 × 106 CFU/g milk, respectively. Milk was heat treated to target temperatures of 63 ± 0.5°C, 65 ± 0.5°C, 67 ± 0.5°C, 68 ± 0.5°C, or 71 ± 0.5°C in five trials. The D values calculated for S. senftenberg 775W ranged from 4.6 at 63°C to 0.17 at 71 °C with z values of 5.0 to 6.7. The D values calculated for L. monocytogenes Scott A ranged from 8.4 at 63°C to 0.19 at 71°C with z value of 4.8 to 6.1. Concomitantly, ALP concentration was monitored using a fluorometric assay. The inactivation rates of the test microbes were greater than or equal to that of alkaline phosphatase over the temperature range tested. The fluorometric assay exhibited excellent accuracy, precision, reproducibility, and repeatability under the test conditions. Viable test pathogens were isolated from milk samples with ALP levels corresponding to legal pasteurization requirements of < 500 milli-Units/L ALP activity assayed fluorometrically (=1.0μg phenol per mL/15 min) when inoculated with 1 × 106 CFU/g milk.