Current detection methods for paper-based analytical devices (PADs) rely on spectroscopic and electrochemical properties, which place special requirements on the analyte or need analyte labeling. Here, ion-transmission mass spectrometry (MS) was proposed for coupling with PADs to enable rapid in situ MS analysis of the sample on paper. The sample was analyzed directly on paper via analyte ionization by ions transmitted through the paper, generated by a low-temperature plasma probe. Prior to MS analysis, the sample can be separated by paper electrophoresis or by paper chromatography, among a variety of other features offered by PADs. The versatility of this technique was demonstrated by MS analysis of a paper microarray, a mixture of amino acids, and whole blood doped with drugs on PADs.