The presentation gives insight to the modern tools of radiochromatography in preclinical and clinical pharmacokinetic and drug metabolism studies. The essential pharmacokinetic and drug metabolism studies of different species (mouse, rat, dog, rabbit and human) provides information to the final drug registration process. The highly sensitive (pg/ml, fg/ml, at/ml) and selective hyphenated techniques (LC/Triple Quad-Jet Stream-ESI-MS and GC/MS-MS, etc.) required for the pharmacokinetic studies had replaced the conventional methods of detections such as GC and HPLC. In the course of drug development radioactive isotope (beta and gamma single and/or double source)-labeled (3H, 14C, 99Tc, 131I) pharmacokinetic studies combined with new generation of triple-quad and high resolution MS techniques (LC, CE, OPLC) are of significant importance. A number of related case studies will be introduced. The former Imaging Techniques (DAR, PIT) and the new generation of in vitro – in vivo Imaging Techniques (MALDI Imaging, nanoScan, PET/MRI in animal and human studies) will also be presented. A complex multi-step process will be illustrated from separation, purification, isolation to structure elucidation (GC-MS, LC-MS/MS, LC-NMR) of minor and major metabolites derived from animal and human biological matrices. The addition of these systems to the off-line and on-line separation and radioactivity detection possibilities of OPLC-DAR/PIT, OPLC-RD, HPTLC-DAR-MS and GC-RD, HPLC-RD and the combined multi-hyphenated techniques, OPLC-DAD-RD-MS/MS, OPLC-DAD-RD-NMR as well as LC-DAD-RD-MS/MS and LC-DAD-RD-NMR resulted in a new, flexible and rapid high-performance complex solution in the metabolism research.