ABSTRACTThe analysis of antimicrobial activity is usually MIC- and minimal bactericidal concentration (MBC)-focused, though also crucial are resistance-related parameters, e.g., the frequency of spontaneous mutant selection (FSMS), the mutant prevention concentration (MPC), and the mutant selection window (MSW).In vitro-determined MPCs, however, are sometimes variable, poorly repeatable, and not always reproduciblein vivo. We propose a new approach to thein vitrodetermination of MSWs, along with novel parameters: MPC-D, MSW-D (for dominant mutants, i.e., selected with a high frequency, without a fitness loss), and MPC-F, MSW-F (for inferior mutants, i.e., with an impaired fitness). We also propose a new method for preparing the high-density inoculum (>1011CFU/mL). In this study, the MPC and MPC-D (limited by FSMS of <10−10) of ciprofloxacin, linezolid, and novel benzosiloxaborole (No37) were determined forStaphylococcus aureusATCC 29213 using the standard agar method, while the MPC-D and MPC-F were determined by the novel broth method. Regardless of the method, MSWs1010of linezolid and No37 were the same. However, MSWs1010of ciprofloxacin in the broth method was narrower than in the agar method. In the broth method, the 24-h incubation of ~1010CFU in a drug-containing broth differentiates the mutants that can dominate the cell population from those that can only be selected under exposure. We consider MPC-Ds in the agar method to be less variable and more repeatable than MPCs. Meanwhile, the broth method may decrease discrepancies betweenin vitroandin vivoMSWs. The proposed approaches may help establish MPC-D-related resistance-restricting therapies.