In this study, the complete CoSYPS Path Food workflow including all steps, namely swab sample enrichment, SYBR®Green qPCR detection of Salmonella spp. and Listeria spp., isolation and confirmation of the detected strain, was validated on beef carcass swabs. To perform the validation, the results of the complete workflow were compared, according to the ISO 16140:2003, with the ISO reference methods for detection, isolation and confirmation of Listeria monocytogenes and Salmonella spp. The results showed that the relative level of detection and the limit of detection of the complete workflow and ISO reference methods are in a range from 2 to 16 CFU/swab for both bacteria. The relative specificity, sensitivity and accuracy identified during this validation were all 100% since the results obtained with the complete CoSYPS Path Food workflow and the ISO reference methods were identical (Cohen's kappa index = 1.00). In addition the complete CoSYPS Path Food workflow is able to provide detection results (negative or presumptive positive) in half the time needed as for the ISO reference methods. These results demonstrate that the performance of the complete CoSYPS Path Food workflow is not only comparable to the ISO reference methods but also provides a faster response for the verification of beef carcasses before commercial distribution.