An integrated evaluation of the pharmacodynamic and pharmacokinetic properties of a drug is essential for successful translation to the clinic. However, cost-effective methods to measure these parameters at the systems level in pre-clinical models are lacking. Here, we introduce a timsTOF and MALDI-2 workflow to evaluate therapeutic activity. The platform combines ion mobility, mass spectrometry-based imaging, absolute drug quantitation across different biological matrices, in vivo isotope tracing, and global metabolome analysis in the adult zebrafish.