Clavulanate potassium (CP) is a classic β-lactamase inhibitor, which is extensively used as a combination drug in antibiotics therapy. As a chiral drug, CP is the only effective stereoisomer revealing prominent activities. To determine the chirality of CP efficiently, we provide an accurate and convenient method based on the comprehensive investigation of circular dichroism spectra and theoretical calculations. The results demonstrate two distinguishable cotton effects in electronic circular dichroism (ECD) and a sequence of cotton effect signs at specific wavenumbers in vibrational circular dichroism (VCD) can be regarded as diagnostic characteristics for the determination of absolute configuration. Additionally, the corresponding spectroscopic appearance of CP was elucidated with the help of theoretical analysis of molecular orbitals, natural bond orbitals and molecular electrostatic potential. The explicit absolute configuration determination of CP facilitates the thorough understanding in the stereochemistry of clavulanate potassium and its derivatives.