A chiral chromatographic analysis using HPLC coupled in series with circular dichroism and optical rotation detectors (HPLC-CD-OR) was applied to racemic trans-stilbene oxide, allowing the on-line measurement of the CD spectra and [α]670 values for both enantiomers in the sample. These measurements used the Gaussian peak model to estimate the concentration of each isomer in the CD and OR cells. Moreover, time-dependent density functional theory (TD-DFT) calculations at the B3LYP/TZVP//B3LYP/VDZ(P) and B3LYP/aug-cc-pVDZ//B3LYP/VDZ(P) levels of theory for CD and OR, respectively, permitted the prediction of both chiroptical properties for each isomer, which in turn could be compared with the corresponding experimental data. In this case, the calculations were consistent in predicting the correct combination of experimental CD bands and [α]670 sign and therefore facilitated the absolute configuration (AC) assignment of the optical isomers. The AC proposed using this methodology was in agreement with independent AC assignments in the literature.