The purpose of present investigation was to understand the interactions involved in complexation of Nelfinavir Mesylate (NM)-a protease inhibitor, used in the treatment of HIV/AIDS with Beta-cyclodextrin (β-CD) and its subsequent effect on its absorption properties and bioavailability. Milling method was used for complexation. The inclusion complexes were characterized by 2D NOESY NMR and ITC studies. The feasibility of findings was further confirmed by using Cerius2 software of Tripos Inc. using Silicon Graphics O2. Pharmacokinetic studies were carried out in rabbits and data was treated by Student’s t Test. 2D NOESY NMR studies showed very intricate behavior showing interactions amongst drug and β-CD molecule as well as amongst β-CD-β-CD molecules. This fact of formation of molecular aggregates was further confirmed by ITC studies. Computer simulation studies further supported the finding of forming shallow complex. The percent relative bioavailability of complex at the dose of 400 mg/kg in rabbits was 185.37 as compared to the plain NM at 400 mg/kg dose. The studies were conducted at low dose of 200 mg/kg of drug in the form of complex in rabbit does not show statistically significant difference in AUC, T 1/2 and Kel. as compared to plain drug at 400 mg/kg of rabbit. © 2007 Springer Science+Business Media, Inc.