Desorption/ionization on silicon (DIOS) tandem time-of-flight (TOF/TOF) mass spectrometry (MS) provides high accuracy and significant fragmentation information, particularly in the characterization of biomolecules. DIOS TOF/TOF offers a high-throughput surface-based ionization platform as well as complete fragmentation through high collision energies. The absence of matrix interference in DIOS allows for the MS and MS/MS analysis of small molecules well below m/z 300. In addition, sample preparation is minimal, and the DIOS chips can be stored and reanalyzed for fragmentation information or accurate mass measurements. The combined benefits of robustness, minimal sample preparation, good sensitivity, high throughput, and sequencing capability make DIOS TOF/TOF a powerful tool for small molecule characterization and protein identification.