Determination of fecal pancreatic elastase content by ELISA is a reliable, non-invasive clinical test for assessing exocrine pancreatic function. Despite the widespread use of commercial tests, their exact molecular targets remain poorly characterized. This study was undertaken to clarify which human pancreatic elastase isoforms are detected by the ScheBo Pancreatic Elastase 1 Stool Test and whether naturally-occurring genetic variants influence the performance of this test. Using recombinantly expressed and purified human pancreatic proteinases we found that the test specifically measured chymotrypsin-like elastases 3A and 3B (CELA3A and CELA3B) while CELA2A was not detected. Inactive proelastases, active elastases and autolyzed forms were detected with identical efficiency. CELA3B gave approximately four times higher signal than CELA3A and we identified Glu154 in CELA3B as the critical determinant of detection. Common genetic variants of CELA3A and CELA3B had no effect on ELISA signal strength with the exception of the CELA3B variant W79R which increased detection by 1.4-fold. Finally, none of the human trypsin and chymotrypsin isoforms were detected. We conclude that the ScheBo Pancreatic Elastase 1 Stool Test is specific for human CELA3A and CELA3B, with most of the ELISA signal attributable to CELA3B.