Abstract: Objective To establish and optimize the Etest method for the in vitro test of drug susceptibility of Bartonella isolates, detect the minimal inhibitory concentration (MICs) of different antibiotics to Bartonella and analyze the drug-resistant spectrum. Methods The optimum experimental conditions of Etest method were established before detection of MICs. This method was compared with the agar dilution method (the gold standard) to evaluate its reliability. The MICs of 22 antibiotics to 7 reference strains of Bartonella were tested by using the established Etest method, and data were analyzed with software SPSS 13.0. Results The optimum conditions for Etest method in vitro test of the drug susceptibility of Bartonella strains were as follows: The optimum medium was soybean trypsin agar medium containing 5% defibrinated sheep blood; the optimal inoculation concentration was 2.0 MCF; the optimum incubation time was 7 days. Bartonella strains were sensitive to 13 antibiotics in vitro (MICin vitro, such as clindamycin. etc. Conclusion Etest method is easy to perform in testing Bartonella strains in vitro, the result is accurate and reliable, and the reproducibility is good, This method can used in in vitro test of drug susceptibility of Bartonella to provide evidence for the development of standards for drug susceptibility of Bartonella.