AbstractMany cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein–protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins ‐ substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel‐lctiv (CELlular biotin‐Ligation for Capturing Transient Interactionsin vivo), uses high‐throughput pairwise proximity biotin ligation for comparing PPIs systematically andin vivo. As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel‐lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel‐lctiv can provide direct information on substrate specificity even for highly homologous proteins.