A new in vitro method was developed to determine the bile tolerance of potentially probiotic lactobacilli. The overnight culture of various lactobacilli strains was inoculated into sterile, half-strength MRS broth supplemented with and without 0.3% (wt/vol) oxgall, buffered with 0.1 M sodium phosphate buffer at a final pH of 7.3, and incubated at 37 degrees C for 12 h under anaerobic conditions. The bile tolerance ability of the lactobacilli strains was expressed as the percentage of the propagation generations of the bacterial cells in the presence of oxgall to those in the absence of oxgall. The bile tolerance ability of 11 strains of 8 Lactobacillus species, including 3 bile salt hydrolase (BSH)-negative strains and 8 BSH-positive strains, was analyzed using the newly developed method and two traditional methods. The results showed that bile tolerance ability of the strains was considerably different depending on the analysis method used. The newly developed method mimics the physiological environment of the human small intestine, and avoids changes in pH and bile salt composition during the incubation period, which are drawbacks of the traditional bile tolerance test methods. Therefore, the analysis method developed in this study is more suitable to screen or compare the bile tolerance ability of lactobacilli strains.