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Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after overnight incubation. MIC determination is used as not only a diagnostic tool in treating bacterial infections for clinicians but also a research method in evaluating the efficacy of an antimicrobial. Multidrug resistance Acinetobacter baumannii (A. baumannii) has emerged in recent years. Accurate determination of resistance by MIC assay is important in coping with this superbug. Here we described a protocol for determining MIC for A. baumannii in hope of assisting researchers and physicians in confirming resistance of clinical isolates correctly.