The apoptotic regulator Bid is a key member of the BH3-only protein family, through which the internal and external apoptotic pathways intercept. In cells, Bid is activated by proteolytic cleavage with caspase 9, which results in the formation of its truncated form tBid. In turn, tBid activates pro-apoptotic members of the Bcl-2 family, such as BAX, leading to the permeation of the outer mitochondrial membrane, considered to be a point of no return in apoptotic programmed cell death. Deciphering the molecular mechanism of this process requires biophysical studies using in vitro models. To amend the latter studies, we present here a redesigned version of Bid, in which the caspase cleavage site was replaced with a thrombin-specific cleavage site. We confirm that the tBid generated by thrombin cleavage has functional activity and can target BAX to mitochondria-like membrane causing their permeabilization. Using a combination of FRET spectroscopy with a site-selective fluorescent label we have probed the membrane-induced separation of Bid fragments after thrombin cleavage. We demonstrate that the latter dissociation and tBid-dependent activation of BAX are modulated by the lipid composition of target membranes, specifically by the presence of cardiolipin, which promotes both processes.