Abstract: This study aimed to evaluate the safety of previously isolated Lactobacillus strains (L. Plantarum L12 and L20) by in vitro and in vivo methods. Antibiotic sensitivity, bile salt hydrolysis activity, D-lactic acid content, and hemolytic activity were analyzed in vitro. Then, a short-term oral toxicity study was conducted in mice, which received 0.2 mL (10⁹ CFU/mL) of experimental bacterial solutions orally for 30 days. The results showed that both strains had good antioxidant activity, high sensitivity to antibiotics, high bile salt hydrolase activity, low D-lactic acid content, and gamma hemolysis. Intragastric administration had no adverse effects on growth, behavior, food intake, organ weight, or histopathological analysis of the treated mice. Compared with control mice, the serum malondialdehyde concentration and liver glutathione content of probiotic-treated mice were statistically different (P<0.05). These results suggest that strains L12 and L20 are nonpathogenic and likely to be safe for human consumption.