Annonaceous acetogenins are bioactive compounds present in the leaves of Annona muricata (soursop) with significant anti-cancer activities. In this study, acetogenin-rich ethanol fraction extracted from soursop leaves was further separated and isolated using a simple open column chromatography technique with silica gel as the stationary phase. The separation procedure consists of three steps, each step employing sequential elution with organic solvents having different polarity as the eluent. Eluted fractions rich in acetogenin compounds were identified using the Kedde reagent which formed a distinctive dark green complex with the unsaturated γ-lactone group present in all of the annonaceous acetogenin compounds. HPLC analysis showed that the initial methanol fraction, as well as isolates from the second and the third isolation steps, contain bullatacin, squamocin, squamostatin-A, and squamostatin-D. Bullatacin produced from this study can be used further as a standard compound for quantitative analysis of other acetogenin compounds contained in soursop leaves extract.