This application discloses a background subtraction-mediated data dependent acquisition method useful in mass spectrometry analysis. The method includes subtraction of background data from precursor ion spectra of a sample in real-time to obtain mass data of component(s) of interest and performs data-dependent acquisition on the component(s) of interest based on the resultant mass data from the background subtraction step. The present invention also encompasses mass spectrometer systems capable of background subtraction-mediated data-dependent acquisition and computer programs adapted for use in the background-subtraction-mediated data-dependent acquisition. The invention thus provides highly sensitive data-dependent acquisition for minor components of interest in a sample.