A simple and rapid pressurized liquid extraction (PLE) and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed for qualitative and quantitative determination of nucleosides, bases and their analogues in natural and cultured Cordyceps. The samples were extracted using PLE. The separation was achieved on a ZORBAX Eclipse XDB-C18 column with gradient elution of methanol and 5 mM aqueous ammonium acetate as mobile phase. Target compounds were identified by characterizing their product ions, precursor ions and retention times. Quantitative analysis of investigated compounds were performed using time programmed selective ion monitoring (SIM) or selective reaction monitoring (SRM) with 10 segments in positive (negative for uridine) ion mode. The results showed that 43 bases, nucleosides and their analogues were detected in Cordyceps, of these 16 compounds were identified. The simultaneous determination of seven nucleosides and six bases in Cordyceps was achieved using PLE and HPLC-ESI-MS/MS method described above, which afforded good linearity, selectivity, precision, recovery, short analysis time as well as LOD and LOQ in the ng/ml range.