Escherichia coli O157:H7 is the most frequent Shiga toxin-producing (STEC) serotype associated with severe humans diseases related to foodborne illnesses. Different STEC-contaminated foods, such as undercooked ground beef, hamburgers, fermented sausages and lettuce, among others, have been identified as sources of contamination in sporadic cases or outbreaks associated with STEC infection. Mass spectrometry (MALDI-TOF MS) is a simple and rapid technique that can be applied for STEC O157:H7 strain detection from a presumptive colony on a culture plate. The technology is based on the analysis of protein spectra resulting from the impact of a laser on a sample crystallized with an organic matrix. The presence of some of the nine biomarker peaks (PBs) (3017m/z, 3083m/z, 3595m/z, 3770m/z, 4012m/z, 4939m/z, 5238m/z, 6037m/z, 6169m/z) and the absence of 9060m/z would allow the detection of STEC O157:H7 according to previous work carried out on the Bruker platform. A comparative analysis with Vitek MS Prime (VMSP) (bioMerieux) was necessary to investigate the presence of the same typical PBs. We tested 163 strains, STEC O157:H7 (n=86), EPEC (n=13), ETEC (n=10), EIEC (n=6), EAEC (n=13), non-toxigenic O157 (n=9) and non-O157 STEC (n=26), and strains of Shigella: flexneri1, flexneri2, flexneri6, dysenteriae1, dysenteriae2, boydii and sonneii. The IVD library was used to corroborate the identification and the RUO database was used to search for PBs. Due to the characteristics of the VMSP equipment and the peak analysis software, differences were found between the PBs detected. It was necessary to use other specific peaks: 5234-5238m/Z and 10163-10168 M/Z for STEC O157:H7 and 5229-5233m/z and 10137 and 10142 m/z for E. coli/Shigella. As a result, a new algorithm for the rapid detection of STEC O157:H7 was developed for the VMSP platform to be used in bacteriology laboratories.