Resistance to apoptosis or cell death is one of the hallmarks of cancer. Venetoclax, a BH3-mimetic approved for the treatment of acute myeloid leukemia and chronic myeloid leukemia, binds to the pro-survival protein BCL2 and initiates apoptosis in the cancer cells. Upon venetoclax binding to BCL2, BH3-only proteins like BIM can be released and activate the key executioner proteins BAK and BAX that permeabilise the mitochondrial outer membrane and release pro-apoptotic factors. BAK and BAX can adopt multiple different conformations, depending on their activation state: a non-activated monomeric conformation or an activated conformation that either leads to the formation of homodimers and permeabilization, or is sequestered by pro-survival proteins like MCL1 or BCLXL. The relative amounts of non-activated BAK and sequestered BAK in cancer cells is an important determinant of whether cells will respond to venetoclax treatment. 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 transition to the active conformation of BAK. 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 or sequestered BAK in those cells. 14G6 is thus an important new tool to assess BAK status in response to anti-cancer treatments and understand mechanisms of resistance to venetoclax.