Due to the toxicity and resistance to treatment with anticancer drugs, various methods are used to improve their efficacy in cancer treatment. In this present study, in order to overcome the limitation of 5-Fluorouracil (5-FU), prodrug strategy has been pursued with using density functional theory (DFT) and molecular dynamics simulation (MDs). The main objective of this study is to examine the mechanisms of drug release from its prodrug form by using the intrinsic reaction coordinate (IRC) calculations. The reaction mechanisms of 5-FU prodrug (EMC-5-FU) in the presence of lactic acid (LA) and water molecule were theoretically studied. The IRC calculations were carried out at the M06-2X/6-311G** level in the aqueous phase through the mechanism of ester hydrolysis to obtain energies, the geometry optimization of all stationary points along the potential energy surfaces (PES), and also to determine the harmonic vibrational frequencies. The results herein presented suggest that three reaction pathways and transition states TS1 to TS2 are involved along the calculated potential energy surface. We found that the drug molecule is released in the third step and this occurs by separation CHO group in the presence of water molecule with the highest energy barrier about 25.9 kcal/mol. Since the carbon nanotubes (CNTs) can act as drug delivery vehicles and deliver anticancer drugs directly to the target cells. Therefore in DFT section, the interaction mechanism of CNTs with 5-FU prodrug is studied by means of DFT method. The atoms in molecules (AIM) and the non-covalent interactions (NCI) between the CNTs and prodrug are used in order to examine the strength and type of interaction between them. The result of negative binding energy values of CNT-prodrug interaction show the stability of these complexes. Our theoretical results show that the more favorable interaction occurs when the prodrug is located inside the carbon nanotube. Furthermore, for design and development of intracellular drug delivery systems, steered molecular dynamics (SMD) simulations was used to investigate the possibility of encapsulated prodrug-CNT penetration through a (1-palmitoyl-2-oleoyl phosphatidylcholine) POPC lipid bilayer. For this purpose, the forces of penetration and the free energies of rupture of POPC bilayer with a Prodrug-CNT were studied. Our simulation results show that encapsulated prodrug-carbon nanotube does not permanently destroy the POPC membrane structure.