A new ionization source based on microwave induced plasma was developed for ambient desorption/ionization. The microwave-induced plasma desorption/ionization source (MIPDI) was composed of a copper Surfatron microwave cavity where a fused-silica tube was centered axially. Stable nonlocal thermodynamic equilibrium plasma was generated in the quartz discharge tube when a microwave at a frequency of 2450 MHz was coupled to the microwave cavity. Analytes deposited on the surface of poly(tetrafluoroethylene) (PTFE) or quartz slide after hydrofluoric acid (HF) etching were desorbed and ionized by the plasma. The performance of the MIPDI technique was validated by the analysis of a variety of chemical substances, polymer compounds, and pharmaceutical drugs using argon or helium as the discharge gas. Protonated [M + H](+) or deprotonated [M - H](-) ions were observed in the positive or negative mode. MIPDI was also used for the analysis of compounds in a complex matrix without any sample preparation. MIPDI was also capable of analyzing liquid samples. The signal-to-noise ratio was 463 in the analysis of 9.2 ng of phenylalanine, and the limit of detection was 60 pg for phenylalanine. MIPDI could desorb and ionize analytes with a molecular weight of up to 1200, which was demonstrated by the analysis of polyethylene glycol 800 (PEG800). MIPDI has advantages of simple instrumentation, relatively high temperature, stability, and reproducibility.