Recent examples have demonstrated that the high-resolution liquid chromatography/mass spectrometry (LC/MS)-based mass defect filtering (MDF) technique was effective in selectively detecting drug metabolites regardless of their molecular weights or fragmentation patterns. The main objective of the current study was to evaluate the general applicability of MDF for drug metabolite detection in typical biological matrices. Mass defect profiles of commonly used biological matrices including plasma, urine, bile, and feces were obtained using an LTQ FT mass spectrometer and were compared with those of 115 commonly prescribed drugs. The mass defect profiles were presented as two-dimensional Y-X plots with the determined mass defects of components on the y-axis versus the corresponding m/z values on the x-axis. The mass defect profiles of the matrices appeared to be similar for each type of matrix across species, yet marked differences were apparent between matrices of a given species. The mass defect profiles of components in plasma, bile, and feces showed significant separation from most of the 115 drugs. The mass defect profiles of urine did not show such clean separation from that of the 115 drugs. The results suggest that MDF has a broad applicability for selective detection of drug metabolites in plasma, bile and feces although the selectivity for detecting urinary drug metabolites is not as good as in the other matrices. In addition, the mass defect profiles of the biological matrices allow for prediction of the effectiveness of MDF for certain applications, and for designing specific MDF windows for selective detection of drug metabolites.