To assess the bioactive properties of guava leaf extract (Psidium guajava L.) in lowering blood glucose, in vivo experiments were designed, including parameters such as the concentration and dose of the extract, the dose of streptozotocin (STZ) used to treat mice, and the time points for administering the extract to STZ-induced mice. The aims of this study were to determine various factors related to the experimental procedure and to evaluate the effects of guava extract in a mouse model. Specifically, the study determined the optimal extract concentrations and examined physiological functions in mice, including body weight changes, oxidative stress through malondialdehyde (MDA) markers in the kidneys and pancreas of mice, blood glucose levels, and insulin and glucose tolerance. At different aqueous extract concentrations ranging from 100 mg/kg body weight (bw) to 800 mg/kg bw, physiological and metabolic abnormalities were observed in mice. There was no significant difference between the mice in the experimental and control groups. A single dose of STZ (100, 150, or 200 mg/kg bw) was used to induce prediabetic conditions in mice, characterized by symptoms such as hyperglycemia, loss of appetite, weight loss, insulin resistance, and impaired glucose tolerance. The results indicate that a dose of 150 mg/kg bw STZ effectively induced these prediabetic characteristics. Additionally, the survival rate of mice in this group was higher than that of mice treated with 200 mg/kg bw STZ.