Freeze-dried chitosan matrices have been developed for sustained release of acetogenins extracted from the leaves of soursop (Annona muricata L.). The chitosan matrices were prepared by freeze drying mixtures of chitosan and acetogenin-rich extract obtained by fractionation of a soursop leaves extract solution. Freeze-dried chitosan matrices were obtained as a homogeneous solid phase, an indication of a complete entrapment of acetogenin extract inside the chitosan matrices. In-vitro release of acetogenins from the chitosan matrices immersed in the simulated gastrointestinal fluids was determined for 24 h. Analysis of acetogenin as total lactones was determined using visible-spectrophotometry with Kedde reagent as the lactone complexing agent. The amount of acetogenins released in the simulated gastric fluid of pH 1.2 is much higher than those released in the simulated intestinal and colonic fluids, with pH values of 6.8 and 7.4, respectively. This observation indicate that the short burst release at low pH medium might be beneficial to enhance further release in the neutral pH medium. Although further experiments are required, a higher acetogenin release in the simulated colonic fluid could be expected due to the presence of B-glucosidase as a colonic enzyme and a long colonic residence time, enhancing the potential of freeze-dried chitosan matrices formulation for treatment of colon cancer.