Atmospheric pressure plasma-based ionization coupled to mass spectrometry is a powerful analytical technique. However, the characteristics of existing ionization sources, especially regarding the reactive species and the effect of the discharge type on the soft ionization, are often not well described. In this work, the active capillary plasma ionization source, which is based on a dielectric barrier discharge, was characterized by optical emission spectroscopy and mass spectrometry. To obtain a better understanding of the requirements for a soft ionization, several reactive species and the energy of the ionization reaction were identified. Charged reactive species such as H3O + , N2 +• , N2H + , NO + , N3 + and N4 +• as well as uncharged species (most probably the excited neutral nitrogen) were all found to contribute to the soft ionization process in dielectric barrier discharge. The energy in the plasma was determined to be in the range from 8-16 eV, based on the ionization energy of nitrogen and the measurements of tungsten hexacarbonyl. Furthermore, not only the type of reactive species was relevant for achieving a soft ionization, it was also crucial that the sample was injected through the inner electrode, which leads to nearly no direct contact with the discharge filaments.Plasma-based ambient desorption ionization mass spectrometry is a fast, simple and direct analysis of various types of samples. 1 Applications have been developed for many different fields such as bioanalytics, environmental sciences and pharmaceutical sciences. 2,3 Since the introduction of ambient ionization by Cooks et al. in 2004 4 , more than 70 different ambient ionization techniques have been reported. 5 A category of it are plasma-based ionization sources, which are able to ionize polar and nonpolar compounds with almost no fragmentation. The various plasma-based ionization sources differ mainly in the type of discharge, the type of excitation and the configuration of the electrodes. 1 The type of discharge (e.g. homogenous or filamentary discharge) depends on the plasma parameters and on the setup of the plasma source. How the type of the discharge determines whether soft ionization is possible has not been systematically studied. Moreover, the ionization process in the plasma is complex and often not completely known due to the many different constituents of a plasma, such as positive and negative ions, electrons, excited states, atoms, free radicals and photons. 6 Despite the large number of reactive species present in a plasma, most of these low-temperature plasma ionization techniques produce molecular ions (e.g. MH + , M +• , M-H -, M -• ) with nearly no fragment ions. The aim of the present study is to reveal the factors responsible for the soft ionization in a plasma-based ionization source for molecular mass spectrometry. This is not only essential for gaining insight into the ionization process but is also for the optimization of the performance of the source in real-world applications. A crucial step toward a better ...