Background: Mothers may experience a period of difficulty adjusting when caring for a child with Down syndrome (DS). Thus, it is thought that using constructive coping mechanisms can reduce stress. Among the many disabilities that negatively affect moms' health and stress levels is Down syndrome. To adopt strategies to regain their equilibrium between resources and disability needs, mothers may need instructional guidelines on how to use available support resources or search for alternative ones. This will help them develop appropriate and effective coping strategies to manage the stresses associated with their children's Down syndrome. This study aimed to evaluate the effectiveness of instructional guidelines on parenting stress and coping mechanisms among mothers of children with Down syndrome. Design: A quasi-experimental design (one group pre/posttest) was used to apply this study. Setting: This study was conducted at the Genes Clinics of Down syndrome affiliated with Sohag University Hospitals. Subjects: A purposive sample from the previously mentioned settings consisted of (100) mothers and their children with Down syndrome. Tools of data collection: Tool (1) A structured interviewing questionnaire to assess children and mothers' personal data and health assessment of children with Down syndrome, Tool (2): Mothers' Knowledge about Down syndrome, Tool (3)  Parent Stress Index Scale, and Tool (4) Coping patterns scale. Results: The study results revealed that there was a highly statistically significant improvement was observed in the studied mothers' knowledge of instructional guidelines implementation. Also, there was a highly statistically significant difference between pre and post-instructional guidelines implementation regarding total level stress level and coping mechanism among mothers having children with Down syndrome, also there was a positive correlation between studied mothers' knowledge, stress, and their coping mechanism pre and post instructional guidelines implementation. Conclusion: It was concluded that instructional guidelines implementation had a positive effect on improving knowledge levels, alleviating stress, and promoting coping strategies among mothers of children with Down syndrome. Recommendations: Conducting educational programs focused on reducing mothers' stress and training parents on how to cope effectively with their disabled children under stress.