A novel technique in mass spectrometry, mass defect filter (MDF), has recently been developed and widely employed in metabolite screening and profiling. This approach depends on the elemental constituents but not the molecular weight or mass fragmentation, which can serve as a useful tool for the screening for natural products. We explored the potential of this technique in the screening and dereplication of natural products and generating natural product profiles. The sensitivity, selectivity, and effectiveness of this technique was determined by the screening of steroidal alkaloids from a methanolic extract of Fritillaria thunbergii, glycyrrhizin analogs, coumarin and flavonoid analogs from a methanolic extract of Radix glycyrrhizae. The capability of this technique to remove matrix or interference ions from a crude extract of Fritillaria thunbergii was examined using peimine as a template. The ion signals of the peimine analogs were buried in the total ion chromatogram before mass defect filter processing. Importantly, after the mass defect filtering processing, the alkaloid analogs were easily identified with a much cleaner ion chromatogram. The major alkaloid analogs that have been previously isolated from Fritillaria thunbergii could be identified as well as some minor alkaloids in the filtered chromatogram, confirming the sensitivity and specificity of this technique. The application of mass defect filter in natural product screening and dereplication was also demonstrated by analyzing glycyrrhizin analogs and coumarin or flavonoid analogs from the extract of Radix glycyrrhizae. The total ion chromatograms after mass defect filtering represent the profiles of individual classes of natural products, which are the best finger-prints of traditional Chinese medicines.