Amoxicillin (AMOX), a penicillin A, belongs to the β-lactam family It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics. Its β-lactamase degradation might be prevented by using a molecular [AMOX:β-CD] complex. The aim of this work was to prepare complexes using two methods and then characterize interactions between AMOX and the native β-CD. The extent of complexation in solution has been evaluated by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and 2D rotating-frame Overhauser enhancement spectroscopy (2D ROESY). Mass changes (TG), calorimetric effects (DSC), and mass spectrometry (MS) were determined on the same sample under identical conditions using the Skimmer coupling system. Skimmer and infrared spectroscopy (FT-IR) were used to characterize the solid state of the binary system. Complexation of AMOX with β-CD was proven by FT-IR, NMR, DSC, and HPLC. The 2D ROESY spectra did not show any dipolar proton interaction of the AMOX with cyclodextrin. The 1:1 stoichiometry of the complex was obtained by HPLC. The stability constant for AMOX with β-CD was determined to be 1,878 M −1. In the [AMOX:β-CD] complex, the phenyl group is included inside the β-CD, and the ionized carboxyl group on the penam ring forms hydrogen bonds with the secondary hydroxyl groups of another β-CD to keep the complex stable. Preparation methods allowed exactly the same complex.