Traditional Chinese medicines (TCMs) are attracting increased global attention because of their potential to provide novel therapeutic agents based on substantial historical records of efficacy in man. Many strategies have been designed for the screening and selection of bioactive compounds from these complex natural products mixtures. Biological fingerprinting analysis (BFA), based on small molecule-biomacromolecule interactions in complex systems, has been applied to screen the multiple bioactive compounds in natural products. Here we review the chromatographic and MS approaches used for BFA of natural products with targeting absorption, distribution, metabolism, elimination and toxicity (ADME/Tox) properties. Such chromatographic methods cover a wide range of applications including liposome, serum proteins, liver homogenate and DNA profiling. MS methods for the characterization of molecular interactions between natural products and target molecules by ESI and MALDI-TOF MS are also discussed.