Background: Blood safety is a crucial health concern, and it is still a problem on a worldwide scale. Pool-testing approach is to combine multiple samples and test them as one pool. A pool-testing strategy can shorten the screening time and increase the test rate, especially during times of inadequate reporting speed and limited test availability. Aim: This research aimed to assess the effectiveness of the pool-testing strategy in Egyptian blood banks and to develop a pooling test to be used in the routine screening of blood donors. We suggest that the use of pooling for blood screening is beneficial, especially in our country, which has a high rate of hepatitis infection and recent extensive government control measures,as well as the importance of potential cost savings in Egypt. Subjects and methods: Sera from 619 volunteer blood donors approaching Suez Canal University Hospital's Blood Transfusion Department, were screened individually for Syphilis and viral infectious markers HBV, HCV, and HIV by ELISA, then pooled into pools of five and retested. Results: The results showed sensitivity, specificity, and accuracy of syphilis and HBV tests were 100% on pooled samples while HCV test showed sensitivity 71%, specificity 100%, and accuracy 99.68%. Conclusion: Our present study demonstrated the importance of a pooling protocol and that a pooling scheme of five samples is helpful for mass screening for syphilis and viral markers in subjects from large populations as blood donors without loss of accuracy.