Using traditional methods, antimicrobial resistance testing (AST) may take several days to obtain the results. Faster methods are therefore urgently needed for timely determination of antimicrobial resistance (AMR). To validate the results of a novel AST method that is in development, we determined the AMR of 8 Escherichia coli and 10 Salmonella enterica clinical, animal, and food isolates to 23 antibiotics and determined time-kill kinetics of ampicillin on a model strain E. coli K-12.