A chiral HPLC analysts combined with time-dependent density functional theory (TD-DFT) calculations was used for the assignment of the absolute configuration (AC) of both enantiomers of the antihistamine drug chlorpheniramine. Circular dichroism and optical rotation detections coupled to the chromatographic system (HPLC-CD-OR) permitted the on-line measurement of the CD spectra and [alpha](670) values, while TD-DFT at the B3LYP/TZVP//B3LYP/VDZ(P) and B3LYP/aug-cc-pVDZ//B3LYP/VDZ(P) levels of theory for CD and OR, respectively, delivered theoretical chiroptical properties in the gas and solution states. Both calculations were consistent in predicting the correct combination of CD spectra and [alpha](670) sign and therefore permitted the absolute configuration (AC) assignment of the optical isomers. The AC proposed using this methodology agreed with a previous X-ray study.