The venom proteome of the black mamba, Dendroaspis polylepis, from Eastern Africa, was, for the first time, characterized. Forty- different proteins and one nucleoside were identified or assigned to protein families. The most abundant proteins were Kunitz-type proteinase inhibitors, which include the unique mamba venom components 'dendrotoxins', and α-neurotoxins and other representatives of the three-finger toxin family. In addition, the venom contains lower percentages of proteins from other families, including metalloproteinase, hyaluronidase, prokineticin, nerve growth factor, vascular endothelial growth factor, phospholipase A2, 5'-nucleotidase, and phosphodiesterase. Assessment of acute toxicity revealed that the most lethal components were α-neurotoxins and, to a lower extent, dendrotoxins. This venom also contains a relatively high concentration of adenosine, which might contribute to toxicity by influencing the toxin biodistribution. ELISA immunoprofiling and preclinical assessment of neutralization showed that polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of D. polylepis venom, albeit showing different potencies. Antivenoms had higher antibody titers against α-neurotoxins than against dendrotoxins, and displayed high titers against less toxic proteins of high molecular mass. Our results reveal the complexity of D. polylepis venom, and provide information for the identification of its most relevant toxins to be neutralized by antivenoms. BIOLOGICAL SIGNIFICANCE: The black mamba, D. polylepis, is one of the most feared snakes in the world, owing to the potency of its venom, the severity and rapid onset of clinical manifestations of envenomings, and its ability to strike fast and repeatedly. The present study reports the first proteomic analysis of this venom. Results revealed a complex venom constituted predominantly by proteins belonging to the Kunitz-type proteinase inhibitor family, which comprises the dendrotoxins, and to α-neurotoxins of the three-finger toxin family. The proteins showing highest acute toxicity were α-neurotoxins, which induce post-synaptic blockade of the neuromuscular junctions, followed by dendrotoxins, which inhibit the voltage-dependent potassium channels. The combination of these two types of toxins in the venom underscores the presence of a dual strategy that results in a highly effective mechanism for prey subduction. This complex toxic arsenal is likely to provide D. polylepis with high trophic versatility. The rapid onset and severity of neurotoxic clinical manifestations in envenomings by D. polylepis demand the rapid administration of effective and safe antivenoms. Preclinical tests showed that an antivenom from South Africa and two antivenoms from India were effective in the neutralization of this venom, albeit differing in their potency. Moreover, ELISA immunoprofiling of these antivenoms against all venom fractions revealed that antivenoms have higher titers against α-neurotoxins than against dendrotoxins, thus underscoring the need to develop improved immunization strategies. The results of this investigation identified the most relevant toxins present in D. polylepis venom, which need to be targeted by antivenoms or other type of inhibitors.