Eurycoma longifolia Jack roots, traditionally used as herbal remedies for increasing libido and better health conditions, contain a high content of quassinoids, the bitter substances and alkaloidal canthinones, indigenous in the Simaroubaceae family that are responsible for the plant pharmacological properties. Amongst the quassinoids, the 20-carbon eurycomanone (EN), 13α(21)-epoxyeurycomanone (EP), eurycomanol (EL) and 13α,21-dihydroeurycomanone (DHY), possess anti-malarial, anti-ulcer, anti-pyretic, anti-osteoporosis, aphrodisiac and anti-infertility properties. In the present study, the chromatographic separation employing macroporous resin Diaion HP-20, Sepabeads SP-700 and SP-207 were evaluated for the enrichment of EN and DHY in E. longifolia methanolic extract. Results showed that among the three macroporous resin, Diaion HP-20 performed the best enrichment and separation characteristics. After single chromatographic fractionation with Diaion HP-20, the content of EN and DHY were increased from 6.13 % and 0.54 % to 30.40 % to 13.96 % w/w, respectively. Meanwhile, a simple and reliable high performance liquid chromatography with ultraviolet detection method was developed and validated for the determination of EN and DHY. Subsequently, the EN-rich and DHY-rich fractions were subjected to pharmacokinetic evaluation in rats. The present finding indicated that DHY was able to achieve a higher peak concentration (Cmax) and total area under the curve (AUC0-∞) compared to those of EN in oral dosing.