One of the major factors contributing to aging and age-related diseases is the well-understood decline in the function of adult stem cells. Quantifying the degree of aging in adult stem cells is essential for advancing anti-aging mechanisms and developing anti-aging agents. However, no systematic approach to this exists. In this study, we developed a method to quantitatively assess the degree of aging in adult intestinal stem cells using a midgut model and two aging markers. First, aging was induced in with the desired genotype, and the anti-aging agent was administered 7 days before dissection. Then, the levels of two intestinal stem cell aging markers found in (PH3 and γ-tubulin) were measured using immunohistochemistry. Finally, fluorescence microscopy was employed to count the number of aging markers and take images, which were analyzed using image analysis software. Using this approach, we quantitatively analyzed the effects of anti-aging agents on the aging of adult intestinal stem cells. This methodology is expected to significantly expedite the development of anti-aging agents and substantially reduce the research costs associated with aging-related studies. Key features • PH3 and γ-tubulin serve as reliable markers for quantitatively assessing aging in intestinal stem cells. • This method for discovering anti-aging agents involves processes such as aging induction, treatment with anti-aging agents, dissection, fixation, antibody staining, and analysis of the results. • Vitamin D, similar to metformin and β-hydroxybutyrate, is an anti-aging agent. • Quantitative analysis of adult stem cell aging will enable the rapid and accurate identification of anti-aging agents and efficacy validation.