Serial twofold dilution methods are most commonly used to identify the antimicrobial activities of antibiotics. This can be achieved by different methods like broth dilution or agar dilution. Though these methods are simple, they can be influenced by various experimental factors and result in discrepancy. The following protocol has been validated for Acinetobacter species, including A. baumannii. It is important to include appropriate control strains to determine the minimum inhibitory concentration values and to compare the experiment results.