Probiotics are living bacteria that, when provided in suitable concentrations, provide health benefits to the host. However, certain antibiotic resistance determinants carried on mobile genetic components, like as tetracycline resistance genes, are frequently discovered in normal probiotic genera and serve as a reservoir of resistance for prospective food or gut pathogens, posing a severe safety risk. Although antibiotic resistance and bacterial pathogenicity evolved on different timelines, they have significant similarities. The link between virulence and resistance is influenced by several genetic mechanisms (e.g., co-selection and compensating mutations), as well as the most common global responses. The interplay of these elements and the resulting biological costs is determined by four major components: the bacterial species involved, virulence and resistance mechanisms, the ecological niche, and the host. Novel diagnostic tools that focus on high-risk clones, as well as rapid testing to detect virulence markers, may assist to address the growing issue of the link between virulence and resistance, which is becoming more advantageous for pathogenic bacteria. The aim of this chapter is to provide the experimental methods adopted for detecting virulence and antibiotic resistance–associated genes in the probiotic bacteria by Polymerase Chain Reaction (PCR).