Background: Probe4Cronobacter test kit is based on the use of a fluorescence-labeled peptide nucleic acid probe (PNA) allied to fluorescence microscopy. A sample is taken after a 24 h enrichment of rehydrated 30 g portions of powdered infant formula (PIF). The method uses ready to use dropper solutions applied directly in the sample. This simple process takes less than 2 h to provide a result. In the presence of Cronobacter species, bright red rod-shaped cells will be visible under a fluorescence microscope. Objective: Probe4Cronobacter validation as a new method for the detection Cronobacter species in Powdered Infant Formula (PIF) under the AOAC Performance Tested MethodsSM (License No. 081702). Methods: The validation study encompassed matrix comparison study, inclusivity and exclusivity testing and robustness studies (stability, kit variation, and ruggedness). Results: The inclusivity and exclusivity testing (50 and 35 strains, respectively) yielded no false negative or false positive results. Probe4Cronobacter was compared to the ISO/TS 22964:2006 in 30 g of PIF samples within method comparison in an unpaired study. A total of 30 samples with both low and high level of inoculation were analyzed by Probe4Cronobacter and compared to the same number of samples screened by ISO/TS 22964:2006. No statistically significant differences between presumptive and confirmed results or between candidate and reference method results were observed. Robustness studies showed a high level of consistency and integrity of the kit when different parameters were varied. The deviation conditions tested did not affect the performance of the kit. Conclusions: Probe4Cronobacter test kit has shown to be a accurate, highly sensitive and robust methods for the detection of Cronobacter spp. in PIF samples.