African cobra (Naja spp.) venom contains toxins dominated by proteins and peptides with inter- and intra-specific variations. There are several FDA-approved drugs from snake venom toxins from other regions, including South America and Asia. Profiling the proteomes of medically important African cobra venoms from different locations will aid in developing more effective anticancer agents. The venoms of spitting cobras (Naja pallida and Naja nigricincta woodi) and non-spitting cobras of the Uraeus subgenus (Naja anchietae, Naja annulifera, and Naja nivea) were fractionated by reverse phase-high performance liquid chromatography (RP-HPLC). Using label-free LC-MS/MS, the venom toxins were identified and grouped into families based on their relative abundance. Venom cytotoxicity of both crude and fractionated samples was tested in pancreatic carcinoma cell lines (MIA PaCa-2) using the Alamar Blue assay. Cell viability analysis revealed a cytotoxic effect of spitting cobra venoms against MIA PaCa-2 cell lines compared to normal MRC-5 cells. Conversely, venoms of non-spitting cobras showed no cytotoxic activity against MIA PaCa-2 cells. Selected RP-HPLC venom fractions from the spitting cobras revealed that N. pallida Fraction 6 and N. n. woodi Fraction 9 at a minimal level were cytotoxic against MIA PaCa-2 cells. LC-MS/MS data showed that while N. pallida Fraction 6 was dominated by basic phospholipase 2 CM-III and Cytotoxin 2, N. n. woodi Fraction 9 was dominated by basic phospholipase 2 CM-III, basic phospholipase 2 CM-II and Cytotoxin 3. These fractions will be purified and studied to determine the mechanisms behind the underlying cytotoxicity against MIA PaCa-2 cells.