In order to determine isoflavone glycosides (calycosin-7-O-β-d-glucoside and formononetin-7-O-β-d-glucoside) and aglycones (calycosin and formononetin), a simple HPLC method with isocratic elution employing hydroxypropyl-β-cyclodextrin (HP-β-CD) as a mobile phase additive was developed. Various factors affecting the retention of isoflavone glycosides and aglycones in the C18 reversed-phase column, such as the nature of cyclodextrins, HP-β-CD concentration, and methanol concentration, were systematically studied. The results show that HP-β-CD, as a very effective mobile phase additive, can markedly reduce the retention of isoflavone glycosides and aglycones, and the decrease magnitudes of isoflavone aglycones are more than those of their glycosides. The role of HP-β-CD in the developed HPLC method is attributed to the formation of the inclusion complexes between isoflavone glycosides (or aglycones) and HP-β-CD. So, the apparent formation constants of the isoflavone glycosides (or aglycones)/HP-β-CD inclusion complexes also were investigated. Isoflavone glycosides (and aglycones) form the 1:1 inclusion complexes with HP-β-CD, and the isoflavone aglycones/HP-β-CD complexes are more stable than the isoflavone glycosides/HP-β-CD complexes. Finally, the optimized method was successfully applied for the determination of isoflavone glycosides and aglycones in Radix Astragali samples.