Apoptosis, a form of programmed cell death regulated by the BCL2 family of proteins, plays a vital role in early development by removing unwanted cells as well as an ongoing role in preventing cancer. BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain either forms homodimers with other BAX or BAK molecules, or is sequestered by the pro-survival proteins MCL1 or BCLXL. The relative amounts of non-activated BAK and sequestered activated BAK in cancer cells is an important determinant of whether cells will respond to BH3 mimetic anti-cancer therapy. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformation. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.