Corrosion behaviour of mild steel in 1M H2SO4 with Ampicillin as corrosion inhibitor has been investigated by using weight loss, Potentiodynamic polarization, Electrochemical impedance spectroscopy, Hydrogen permeation and diffuse reflectance spectroscopic studies. All these techniques reveal that inhibition efficiency increases with increase in the concentration of Ampicillin. Scanning electron microscopy (SEM) was carried out to characterize the surface morphology of the metal specimens. Polarization studies indicated that Ampicillin behaved as cathodic inhibitor. Diffuse reflectance spectroscopy confirmed the adsorption of inhibitor on the mild steel surface obeying Langmuir adsorption isotherm. A chemoffice 3D simulation technique was used to run the quantum mechanical analysis and established correlations between different types of descriptors and measured corrosion inhibition efficiencies of Ampicillin. The quantum chemical analysis substantiates the inhibition efficiencies of the compound determined by electrochemical methods. © 2013 University of Manchester and the authors.