A UPLC method for the determination of acetoside in Macfadyena unguis-cati was established. Single factor and orthogonal experiments for acetoside extraction from M. unguis-cati were designed. Macroporous resins were screened for the enrichment of acetoside and the elution procession was determinated. The UPLC conditions: HSST3 column (2.1 × 100 mm, 1.8 μm), mobile phase 0.1% HCOOH(A)–CH3CN(B), DAD detector at 310 nm, flow rate 0.3 mL·min-1, column temperature 25 ℃, elution process: 90%A-5 min, 75%A-7.5 min, 30%A-9 min, 90%A. The results showed that the calibration curves of acetoside was in a good linearity over the range of 0.25—100 ng under the experiment condition and the regression equation was Y=89.002X-4.8275 (R=0.9999). The best extraction solvent was 70% ethanol according single factor experiment and the optimum extraction conditions were 70% ethanol, solid-liquid ratio of 1∶10 and 12 h extraction time through orthogonal experiments. The most suitable macroporous resin for acetoside enrichment was HP-20 through static and dynamic adsorption and desorption experiments. The enrichment process by HP-20 resin was gathering 4 BV of the 40% ethanol elute after 4 BV of elution by 15% ethanol.