Identifying the molecular mechanisms underlying antibiotic resistance is important, as it can reveal key information on the mode of action of a drug and provide insights for the development of novel or improved antimicrobials. Here, we describe an agar-based method for the selection of bacterial strains with increased antibiotic resistance, and how the increase in resistance can be confirmed by a spot-plating assay. As a specific example, we describe the selection ofStaphylococcus aureusstrains with increased resistance to oxacillin; however, the protocol can be easily adapted and used with other bacteria and antibiotics.