The cyclic nucleotide phosphodiesterase (PDE) plays an important role in regulating the levels of second messenger molecules cAMP and cGMP. Various PDE inhibitors have been successfully developed into drugs for targeted diseases. In addition, PDE inhibitors can also be found in different foods and natural medicines. In this study, ultrafiltration liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry (ultrafiltration LC-DAD-ESI-IT-TOF-MS) was applied to screen PDE inhibitors from the roots of Ilex pubescens Hook. et Arn. As a result, 11 major compounds were identified in I. pubescens roots, with nine compounds as potential PDE inhibitors, among which five were further confirmed to be active against PDEI and PDE5A dose-dependently in vitro, with ilexsaponin A1 and ilexsaponin B2 being the strongest. HPLC quantification of these bioactive compounds suggested that they are major components in the plant. The results demonstrate that ultrafiltration LC-DAD-ESI-IT-TOF-MS is an efficient method for rapid screening of PDE inhibitors from natural medicines.