Advancements in molecular technology have revolutionized the detection of phytopathogenic bacteria, with specific amplification of target DNA fragments emerging as a powerful approach, especially for quarantine bacteria. Current conventional PCR protocols for Bacterial Leaf Spot (BLS) xanthomonads primarily focus on species differentiation or the detection of specific species, lacking a comprehensive and validated method for all BLS xanthomonad lineages. The conventional PCR protocol optimized with designed newly primer pair Bs LepA F/ Bs LepA F followed by validation for specificity and sensitivity assays conducted in this study. The protocol was validated using target BLS Xanthomonas strains from major tomato-growing regions in India and non-target bacterial strains belongs to different genus. The detection assays from pure bacterial suspension and threshold detection efficiency from artificially infected leaf samples demonstrating high specificity and sensitivity. Furthermore, the protocol's effectiveness to detect naturally infected plant samples and multiplex with other tomato bacterial pathogens make it a significant tool for agricultural disease control. This research advances molecular detection techniques and provides practical solutions for early identification and management of BLS xanthomonads, protecting tomato and pepper crops from this destructive disease.