Pooled samples play a vital role in screening infections by efficiently utilizing resources and reducing testing costs. They enable the detection of infections even with limited resources while maintaining diagnostic accuracy, making them invaluable for less developed countries. 7-Alpha-hydroxy-4-cholesten-3-one (C4) serves as a valuable marker for screening bile acid loss syndrome due to its correlation with the rate-limiting enzymatic step catalyzed by CYP7A1. This study presents a protocol for screening for the syndrome using C4 values and pooled samples. The protocol involves a two-step approach, where the diagnostic test is initially conducted on a pooled sample. If the pooled sample tests are positive, subsequent individual tests are performed, otherwise, no further tests are required. The use of pooled sample sizes ranging from 2 to 20 leads to an expected reduction of up to 70% in the number of tests compared to individual sample testing strategies. The diagnostic accuracy of the strategy assessed by the area under the receiver operating characteristic (ROC) curve is at least 0.90 for all pooled sample sizes.