An automated flow injection analysis (FIA) mass spectrometry system (AutoScan) was developed to allow rapid unattended determination of optimal conditions during mass (ms) and tandem mass spectrometry (ms/ms) on new chemical entities (NCEs) arranged in 96-well plates. The 96-well plate is placed on the deck of a modified Gilson Multiprobe autosampler for injection into a PE Sciex API 2000 triple quadrupole mass spectrometer. A customized software interface is used to create the necessary scan experiments by associating each 96-well plate of NCEs to be scanned with an index file containing data on the identity of each analyte and its expected molecular weight. Analytes are injected four at a time into a custom injection manifold and conventional mass spectra are acquired in both polarities (+/-) using an alternating positive/negative Q1 scan function. The software determines the optimal polarity and definitive precursor ion for all analytes and uses the results to build the injection sequence for product ion scanning. The samples are automatically re-injected under MS/MS conditions, and product ion scans that loop among different collision energies are collected for each analyte. The resulting data are processed automatically and the optimal MS/MS transitions for each analyte are selected. A color-coded graphical interface facilitates data review. Any unusual ion transitions or transposition errors made during plate preparation are noted and corrected. Complete MS and MS/MS conditions are obtained for 96 compounds in about one hour and the resulting data are available for download as sample control injection sequence files.