Data-dependent acquisition (DDA) is the most common method used to control the acquisition process of shotgun proteomics experiments. While novel DDA approaches have been proposed, their evaluation is made difficult by the need of programmatic control of a mass spectrometer. An alternative is in silico analysis, for which suitable software has been unavailable. To meet this need, we have developed MSAcquisitionSimulator-a collection of Cxe2x80x89++ programs for simulating ground truth LC-MS data and the subsequent application of custom DDA algorithms. It provides an opportunity for researchers to test, refine and evaluate novel DDA algorithms prior to implementation on a mass spectrometer.