The widespread misuse and overuse of antibiotics in the agrifood sector have significantly contributed to the emergence of antibiotic-resistant bacteria, which pose a serious public health threat. Furthermore, because antimicrobials often share mechanisms of action, there is an increased risk of developing cross-tolerance. These phenomena underscore the need for an in-depth study of the molecular mechanisms behind resistance. In this context, the deliberate generation of antibiotic-resistant variants (RVs) provides a valuable tool for investigating the mechanisms of resistance acquisition and maintenance. This chapter outlines a step-by-step protocol for obtaining RVs through exposure to progressively increasing doses of antibiotics. Additionally, it presents a methodology for the detection and annotation of mutations in the RVs. By mapping and analyzing these mutations, researchers can better predict the role of specific genetic changes in the development of different forms of antibiotic resistance, offering insights into potential strategies for combating this global challenge.