A standardized protocol for repeated social defeat stress in mice is described, which involves repeatedly subjecting C57BL/6J mice to social defeat by a larger, aggressive CD-1 mouse. This protocol results in the development of a depressive-like syndrome, characterized by social avoidance and other behavioral and physiological changes. The protocol consists of three stages: selection of aggressive CD-1 mice, agonistic social confrontations, and confirmation of social avoidance in subordinate C57BL/6J mice. The automated detection of social avoidance increases throughput, reproducibility, and quantitative analysis. The protocol is highly adaptable and typically takes 3–4 weeks to complete. Social defeat stress is a valuable model for studying depression-like behavior, as it has strong etiological, predictive, discriminative, and face validity. It is particularly useful for examining individual differences in depression and anxiety-related behaviors. Social defeat stress is also effective in identifying compounds with rapid antidepressant effects compared to traditional selective serotonin reuptake inhibitors (SSRIs). The model has been shown to respond only to chronic, not acute, antidepressant treatments, suggesting pharmacological validity. The protocol involves screening CD-1 mice for aggression, followed by repeated social defeats of C57BL/6J mice. Social avoidance testing is conducted using an open-field arena with automated tracking. The protocol is designed to be flexible and can be adapted for various molecular and behavioral studies. It is important to note that the model may require a moderate financial investment, larger space, and time for each run. The protocol is validated through multiple stages, including the selection of aggressive CD-1 mice, the social defeat process, and social avoidance testing. The model has been shown to produce consistent results and is useful for studying the molecular basis of affective disorders. The protocol is also applicable for studying the effects of various treatments on depression-like behaviors. The model is widely used in research and has been shown to be effective in identifying new therapeutic interventions for depression. The protocol is described in detail, including the materials, equipment, and procedures required for its implementation. The results of the protocol are used to analyze the behavioral and physiological changes associated with social defeat stress. The protocol is a reliable and reproducible method for studying depression-like behaviors in mice.A standardized protocol for repeated social defeat stress in mice is described, which involves repeatedly subjecting C57BL/6J mice to social defeat by a larger, aggressive CD-1 mouse. This protocol results in the development of a depressive-like syndrome, characterized by social avoidance and other behavioral and physiological changes. The protocol consists of three stages: selection of aggressive CD-1 mice, agonistic social confrontations, and confirmation of social avoidance in subordinate C57BL/6J mice. The automated detection of social avoidance increases throughput, reproducibility, and quantitative analysis. The protocol is highly adaptable and typically takes 3–4 weeks to complete. Social defeat stress is a valuable model for studying depression-like behavior, as it has strong etiological, predictive, discriminative, and face validity. It is particularly useful for examining individual differences in depression and anxiety-related behaviors. Social defeat stress is also effective in identifying compounds with rapid antidepressant effects compared to traditional selective serotonin reuptake inhibitors (SSRIs). The model has been shown to respond only to chronic, not acute, antidepressant treatments, suggesting pharmacological validity. The protocol involves screening CD-1 mice for aggression, followed by repeated social defeats of C57BL/6J mice. Social avoidance testing is conducted using an open-field arena with automated tracking. The protocol is designed to be flexible and can be adapted for various molecular and behavioral studies. It is important to note that the model may require a moderate financial investment, larger space, and time for each run. The protocol is validated through multiple stages, including the selection of aggressive CD-1 mice, the social defeat process, and social avoidance testing. The model has been shown to produce consistent results and is useful for studying the molecular basis of affective disorders. The protocol is also applicable for studying the effects of various treatments on depression-like behaviors. The model is widely used in research and has been shown to be effective in identifying new therapeutic interventions for depression. The protocol is described in detail, including the materials, equipment, and procedures required for its implementation. The results of the protocol are used to analyze the behavioral and physiological changes associated with social defeat stress. The protocol is a reliable and reproducible method for studying depression-like behaviors in mice.