The purpose of this study is to analyze the mediating effect of shoulder pain, shoulder disorder, and depression in the relationship between stress and health-related quality of life in middle-aged women using a serial mediation model. For this study, data was collected from middle-aged women aged between 35 and 64 living in Seoul, South Korea, from May 13, 2021 to May 23, 2021. Data regarding stress, health-related quality of life, shoulder pain, shoulder disorder, and depression were collected using a self-reported, structured survey. SPSS macro process (Model 6) was used, and the relationship between stress and health-related quality of life of participants were analyzed using serial mediation analysis using shoulder pain, shoulder disorder, and depression as variables. From the results of the study, it was observed that stress has a statistically direct impact on health-related quality of life. On performing serial mediation analysis, shoulder pain, shoulder disorder, and depression were found to be statistically significant, which predicts the relationship between stress and health-related quality of life; the variables showed an explanatory power of 33% for influencing the quality of life. Therefore, the relationship between stress and health-related quality of life was found to be partially mediated by shoulder pain, shoulder disorder, and depression. This study suggests the need for healthcare workers to develop a method, such as exercise intervention programs based on various degrees and types of physical activity, for improving the health-related quality of life and reducing stress caused by shoulder pain, shoulder disorder, and depression in middle-aged women.