Prime cost determinations can increase managerial effectiveness in budget allocation and strategic planning. This study was designed to calculate the prime cost of clinical laboratory services in Tehran Valiasr Hospital using the activity-based costing (ABC) model. The highest costs were for human recourses (44%) and the lowest for energy (5%). The largest proportion of activities (97%) was by specialists, reflecting the importance of human recourses in prime costs. The highest prime cost was for urinalysis (11% of tests) and the lowest for sodium determination (21% of tests), which demonstrates that prime cost decreases as service frequency increases. The average estimated prime cost was 63% higher than the fees established by the Ministry of Health and Medical Education. The results show that the Tehran Valiasr Hospital laboratory faces a budgetary deficit. The prime cost of services can be reduced by improving human recourse management and standardization of resource consumption.