Standardization in behavioral testing is essential for reproducibility and translational relevance in preclinical research. Overhead video monitoring systems, commonly used to assess spontaneous behavior in rodents, can vary significantly in test duration and interpretation, which limits cross-study comparisons. This study aimed to define optimal short-term overhead video monitoring durations and provide behavioral references across age and sex in naïve C57BL/6 mice. We conducted open-field assessments with juvenile and adult mice of both sexes, analyzing key behavioral parameters relating to mobility, exploration, and anxiety/vigilance. Our findings demonstrate that behavior varies significantly with age and sex. Juvenile mice exhibited exploratory behaviors and resting behaviors, while adults had increased anxiety-like behaviors; female mice showed more complex and variable behavioral patterns compared to males. The 2.5-minute sessions revealed stable and statistically significant differences in male behavior, suggesting that short-term monitoring can be both efficient and informative when applied appropriately. However, longer sessions (?10 minutes) were required to fully capture behavioral variability in females. This study provides empirical guidance for selecting test durations and interpreting behavioral data in overhead video monitoring. The results support the implementation of sex- and age-appropriate protocols to improve the consistency and interpretability of behavioral outcomes. These insights contribute to the broader goal of standardizing preclinical mobility and pain assessments, thereby enhancing the rigor and utility of rodent models in osteoarthritis and related fields.