Tomatidine is an aglycone of α-tomatine, a glycoalkaloid present in tomato plants, and has muscle atrophy inhibitory effect and anticancer activity. Tomatidine-Rich Tomato Leaf Extract powder (TRTLE) contains 60% tomatidine, which is converted to tomatidine by acid hydrolysis after extracting α-tomatine from tomato leaves. The purpose of this study was to evaluate the safety of TRTLE by conducting a series of toxicity studies in mice and bacteria to support its safe food use. Single-dose and 90-day repeated-dose toxicity study were conducted in 6-week-old ICR male and female mice to calculate the LD₅₀ and non-toxic dose (NOAEL) of TRTLE. In the single-dose toxicity study, a single oral dose of 667, 2,000, and 5,000 mg TRTLE/kg body weight (bw) was administered, and in the 90-day repeated-dose toxicity study, 133 mg TRTLE/kg bw was administered orally daily. In addition, the Ames test was performed with Salmonella Typhimurium and Escherichia coli to determine the genotoxic activity. The single-dose toxicity study indicated the LD₅₀ was 833 mg TRTLE/kg bw (tomatidine equivalent: 500 mg/kg bw). In the 90-day repeated-dose toxicity study, no abnormalities due to TRTLE were observed in each laboratory test, including general symptoms, body weight changes, hematology, urinalysis, and histopathological examination. In the Ames test, TRTLE was confirmed not to be mutagenic with or without metabolic activation. Based on these data, the NOAEL in mice was determined to be 133 mg TRTLE/kg bw (tomatidine equivalent: 80 mg/kg bw).