To provide a useful screening method for selecting probiotics, we compared the pH and bile resistance of four strains of Lactobacillus acidophilus, KCTC3140, KCTC3146, KCTC3154, and KCTC3179, isolated from a rat, pig, chicken, and human, respectively. When we compared the pH resistance of these strains at pH 2, 3, 4, 5 and 7, we found that L. acidophilus isolated from the rat, chicken, and pig showed little or no decrease in viable cell numbers, except at 240 min, whereas the numbers of L. acidophilus KCTC3179 from the human decreased significantly. All four strains were slightly suppressed over time and showed bile resistance, even at 3%. At 5% oxgall, the number of KCTC3179 rapidly decreased at 30 min. These results indicate that lactic acid bacteria selected for probiotic use should be screened at pH 2 for 120 min and/or at an oxgall concentration of 5% for 30 min.