Objective:This study aimed to optimize the determination conditions of pantothenic acid content in infant milk powder by microbiological method and provide test data for the subsequent revision of the national standard method for pantothenic acid detection.Methods:The issues covering the determination of pantothenic acid by the national standard method were analyzed, and the key steps in the detection process, including bacterial solution preparation, pretreatment, and absorbance determination, were emphatically studied.Results:Lactobacillus plantarum from ATCC and CICC can be used to determine pantothenic acid. The best results were obtained by directly inoculating 80% bacterial solution concentration into the test tube, or adding 5 mL bacterial solution to 400 mL sterilization test medium. The results of direct extraction were higher than that of hydrolysis. The relative deviation of enzymole-plate and UV spectrophotometry was less than 5%. The recovery rate was 96.9%, and the relative standard deviation (RSD) was 4.95%.Conclusion:The optimization of the key steps in the determination of pantothenic acid by microbiological method can simplify the operation process and improve the accuracy of the samples, and the optimized conditions can be used as a reference for the subsequent revision of the National standard method for pantothenic acid determination.