Infertility has emerged as a global health concern, impacting around 8%-12% of couples during their reproductive years. Due to limitations in obtaining human biological samples, mouse models have been widely used for investigating gene functions. Fertility assessment in mouse models is a critical component in reproductive biology for studying gene function and elucidating mechanisms of reproductive disorders. However, natural mating observation of mice may yield inconsistent results, especially in the absence of standard guidelines, prolonged experimental cycles, and operational complexity. This protocol establishes a comprehensive breeding strategy for evaluating murine fertility through systematic vaginal plug monitoring and litter size quantification within defined timeframes. Key steps include (1) standardized male-female pairing protocols, (2) daily vaginal plug inspection, and (3) longitudinal tracking of pregnancy outcomes. This protocol presents a straightforward and easily implementable protocol for mouse mating cage setup and statistical analysis, enabling reliable fertility assessment under natural breeding conditions. Key features • Standardized natural mating protocol combining vaginal plug monitoring (daily) and litter size tracking. • Time-optimized workflow completes fertility phenotyping in 2 months. • An easy and custom-constructed vaginal plug inspection tool for optimized vaginal plug inspection.