Acute myeloid leukaemia (AML), a cancer of the myeloid line of blood, is the most common myeloid leukaemia and is responsible for over 10,000 deaths each year. With chemotherapy being the only available treatment for AML patients to date, novel treatments are desperately needed. Smac mimetics are currently in clinical trials for the treatment of various cancers. These drugs antagonize inhibitor of apoptosis proteins (IAPs) and simultaneously induce TNF secretion to render cancer cells sensitive to TNF induced killing. We have previously shown that the combination of a Smac mimetic and p38 inhibitor achieves synergistic killing in AML (Lalaoui et al., Cancer Cell 2016). Furthermore, the inhibition of the MAPK p38 is able to overcome Smac mimetic resistances observed in some AMLs. The exact molecular mechanism by which p38 inhibitors increase Smac mimetic mediated killing is still unknown. Using a phosphoproteomic approach we therefore seek to identify mediators that are involved in the synergistic effect between Smac mimetics and p38 inhibitors. To achieve this, we have investigated time-dependent changes in the phosphoproteome of murine bone marrow-derived macrophages (BMDMs) treated with a Smac mimetic alone or in combination with a p38 inhibitor. We used a label-free quantitative phosphoproteomics approach whereby phosphopeptides were enriched using magnetic Fe-NTA beads. Subsequent analysis of our quantitative phosphoproteomics data revealed and enrichment of kinases with known roles in the observed synergism. Moreover, we have uncovered a number of phosphorylated proteins that potentially represent novel mediators of the mechanisms behind the synergistic killing of AML cells via Smac mimetics and p38 inhibitors. This research paves the way for new therapeutic targets in the treatment of AML.