The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6μm−2 to ≃0.1μm−2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.