Cholera is one of the most serious diarrheal diseases, responsible for causing at least seven pandemics in the world. Among the virulence factors associated with this disease is cholera toxin, encoded by the ctxA gene and the pilus coregulator of toxin encoded by the tcpA gene, plays an important role in pathogenicity. objective. To validate a multiplex pcr technique for the diagnosis of toxigenic Vibrio cholerae based on the detection of Vc-m, ctxA and tcpA genes. methods. The validation of the technique was carried out in the National Institutes of Health’s Laboratory of Enteropathogens, using conventional pcr (Vc-m) and pcr multiplex (ctxA-tcpA) assays, and the standardization of multiplex pcr with three targets (Vc-m/ctxA/tcpA), validation was performed with 51 clinical isolates of toxigenic Vibrio cholerae (ctxA) and 31 non-ctxA-producing strains of different genera. The range of work, selectivity, robustness, sensitivity, specificity, positive predictive value and positive predictive value of the technique were determined. results. The methodology was validated, obtaining a detection range ranging from 10-0 dilution to 10-2. Robustness was optimal when modifying different variables. We obtained 100% inclusivity, exclusivity, analytical precision, positive predictive value and negative predictive value, as well as sensitivity and specificity. conclusions. The standardization and subsequent validation of multiplex pcr (Vc-m/ctxA/tcpA), performed by different operators, corroborates the parameters considered, confirmed the repeatability and reproducibility of the technique, thus providing a fast, safe and selective for the identification of toxigenic Vibrio cholerae isolates from clinical isolates after primary isolation.