Effective monitoring and control measures are essential to reduce the prevalence of _Salmonella_ in food sources and hence the number of human cases of salmonellosis [4]. Eggs and poultry meat are the main sources of _Salmonella_ infections in humans [5,6]. Control of _Salmonella_ in poultry is therefore a priority for the industry and public health authorities. However, _Salmonella_ infections in poultry caused by serotypes with a broad host range, such as Typhimurium, Enteritidis or Indiana, are usually asymptomatic and are therefore difficult to detect [7]. Nonetheless, they can transmit salmonellosis to humans via the food chain. Strategies to reduce its prevalence include biosecurity measures, regular testing, and vaccination programs [8,9]. The use of live attenuated vaccines is an effective approach to controlling _S. enterica_ subsp. _enterica_ sw. Typhimurium infections in poultry [10]. Primun Salmonella T vaccine strain ST CAL16 Str+/Rif+/Enr- (Calier, Leon, Spain) is a live attenuated vaccine that can be used in breeding hens, laying hens, and broilers to reduce the prevalence of _S. enterica_ subsp. _enterica_ sv. Typhimuriumium in eggs and poultry meat. While live attenuated vaccines are effective in reducing disease incidence, they can complicate the diagnostic process because they can be misidentified as wild virulent strains. Consequently, detection methods that can distinguish the vaccine strain from wild strains are required for the safe use of a live vaccine in the field. Wild and Primun Salmonella T vaccine strains can be distinguished by their antimicrobial resistance profiles. Unlike the field strains, the vaccine strain is sensitive to enrofloxacin and resistant to streptomycin and rifampicin. Therefore, the initial method for differentiating Primun Salmonella T vaccine strain from wild strains relied on antibiograms. However, this approach presents significant limitations as it depends on the stability of resistance patterns among circulating wild strains. Wild strains may eventually show the same antibiotic resistance profile as the ST CAL16 Str+/Rif+/Enr- strain [11]. In fact, antibiotic overuse has led to an increase in antimicrobial resistance [12]. Consequently, the antibiogram method could fail to differentiate the vaccine strain from wild strains. The development of a more accurate and easy-to-use technique for differentiating infected from vaccinated animals (DIVA) is essential to ensure a rapid and appropriate response to detected infections and the effectiveness of vaccination programs. Indeed, incorrect characterisation of wild strains as vaccine strains could lead to a salmonellosis outbreak, while incorrect characterisation of vaccine strains as wild strains could lead to inappropriate slaughtering of animals and important economic losses. DIVA assays typically rely on the differentiation of specific antigens, genetic markers, or phenotypic traits unique to the vaccine strain. Molecular approaches, particularly those based on polymerase chain reaction (PCR) technologies, have emerged as the most promising tools due to their superior sensitivity, specificity, and turnaround time compared to conventional microbiological methods. A duplex qPCR assay was specifically designed for the accurate and reliable differentiation of wild _S. enterica_ subsp. _enterica_ sv. Typhimurium strains (SaITypm assay) and the Primun Salmonella T vaccine (PriSal-T assay) from isolated colonies. This qPCR-based DIVA assay has the advantage of being significantly faster than antibiogram-based techniques; however, it is important to ensure its sensitivity and specificity. In this study, the SaITypm&PriSal-T qPCR Duplex kit was validated by assessing its analytical and diagnostic performance.