Glycerolipidic prodrug is an interesting concept to enhance lymphatic absorption of polar drugs intended to oral delivery such as didanosine (ddI). In order to improve ddI bioavailability, two didanosine glycerolipidic prodrugs, the phosphorylated (ProddIP) and the non-phosphorylated derivatives (ProddINP) were synthesized to follow triglyceride metabolism. The biomimetism approach of these prodrugs has been studied in vitro at two steps. First, liposomal formulation of each prodrug was incubated with a lipolysis model based on pancreatin and analysed using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). These experiments evidenced that both didanosine prodrugs were recognized by the lipases; as expected, they were cleaved at both positions sn-1 and sn-3 of glycerol. ProddIP was metabolised twice more rapidly than ProddINP suggesting an implication of some phospholipases in ProddIP degradation. Secondly, the detection of dideoxyadenosine triphosphate (ddA-TP) into HIV-1 infected cells after their incubation with ProddINP loaded liposomes evidenced their ability to release ddI that could penetrate into the cells and be metabolised by intracellular kinases. These results confirmed that the synthesized glycerolipidic prodrugs of didanosine could be investigated for a biomimetic approach with final aiming of increasing the drug oral bioavailability by enhancing intestinal absorption.