<p>Dimerization of transmembrane (TM) proteins is a fundamental process in cellular membranes, central to numerous physiological and pathological pathways, and increasingly recognized as a promising therapeutic target. Although often described as a simple two-state transition from monomers to dimers, the process following monomer diffusion—referred to as post-diffusion dimerization—is likely more intricate due to complex inter-residue interactions. Here, we present a single-molecule tweezer platform that directly profiles these post-diffusion transitions during TM protein dimerization. This approach captures reversible dimerization events of individual TM dimers, revealing previously hidden intermediate states that emerge after monomer diffusion. By integrating measurements of intermediates, kinetics, and energy landscapes with molecular dynamics simulations, we delineate the dimerization pathway and dissect how residue interactions and lipid bilayers influence the process. Furthermore, our platform allows for the targeted analysis of localized perturbations—such as those induced by peptide binding or site-directed mutagenesis—demonstrating its utility for probing the mechanisms of TM dimer-targeting therapeutics at single-molecule resolution.</p>