Introduction: Exercise is an important step in preparation and implementation of disaster risk programs. The aim of this research was the development and assessment of an operational exercise for disaster preparedness in southeast Iran. Methods: A descriptive cross-sectional study was used to evaluate a 2-day exercise for a level 4 (L-4) scale mock disaster conducted in southeastern Iran in 2017. Seven medical sciences universities participated in the mock disaster exercise: Kerman, Rafsanjan, Bam, Jiroft, Zahedan, Zabol, and Iranshahr medical sciences universities. In total, 990 personnel participated. The disaster preparedness was assessed using a tool designed by the exercise planning team. Results: The present research reveals that the universities of medical sciences in the southeastern hub of Iran are properly prepared for emergencies and disasters. This included the Disaster Medical Assistance Teams and the response operational comprehensive plan for emergencies and disasters. The research findings indicated a preparedness score of 79.8%. The mean arrival time at exercise area (Jiroft City) was 4 hours 35 minutes despite the wide area of Kerman and Sistan and Baluchistan provinces. Personnel safety and security, rapid assessment, communications, logistics, warnings, and news confirmation functions showed higher levels of preparedness. Conclusion: The Joint Commission on Accreditation of Healthcare Organizations reported that organizations must perform the performance preparedness exercises on different levels and areas twice a year to be properly prepared for responding to emergencies and disasters. This study can be considered a suitable standard guide for health care organizations to execute exercises for the maintenance and promotion of their preparedness for properly responding to emergencies and disasters at L-4.