Measuring protein-protein interactions using purified proteins in vitro is one of the most frequently used approach to understand the biochemical and mechanistic details of cellular signaling pathways. Typically, affinity tags are genetically fused to proteins of interest, and they are used to capture and detect them. However, in some cases, fusion of bulky affinity tags might present a significant limitation in these experiments, especially if the regions in close proximity of tags are involved in protein-protein interactions. Here, we present a step-by-step protocol for an alternative approach that involves reversible biotinylation of purified proteins using a simple chemical-conjugation of cleavable biotin moiety. Biotinylated proteins can be directly used as bait for selective immobilization on solid support for measuring protein-protein interactions. Furthermore, biotinylation of protein of interest also allows specific detection in standard biochemical assays. This simple, straightforward and modular protocol can be directly adapted and applied to facilitate the detection of novel protein-protein interactions as well as measuring apparent affinities of such interactions.,? 2020 Elsevier Inc. All rights reserved.