A robust method for in vitro metabolite generation and facile sample preparation on analytical HPLC was established for rapid structure determination of microgram-level drug metabolites by using high-field NMR equipped with a cryoprobe. A single 1–5 mL incubation of drug candidate (10–30 μM) in microsomes, hepatocytes, or recombinant drug-metabolising enzymes, typically cytochrome P450s and UDP-glucuronosyltransferases, was used for metabolite formation. Following precipitation of proteins and solvent removal, metabolite mixtures were chromatographed with 5–10 injections onto an HPLC-MS system. Metabolites were collected into a 96-well plate, dried, and reconstituted in deuterated NMR solvents. NMR spectra of isolated metabolites were acquired on a 500 MHz spectrometer equipped with a 5 mm cryogenic probe. The methodology has been successfully employed as an extension of HPLC-MS/MS-based metabolite identification and applied frequently to 0.5–10 μg quantities of metabolite. Most structure determinations were achieved rapidly by 1D ¹H NMR with satisfactory signal-to-noise ratios, whereas some required 2D NMR data analysis. This report describes the method development and metabolite structure determination using the model compound trazodone. In addition to trazodone, a large number of examples from our laboratories have proven that the microgram-level NMR method avoids time-consuming preparative-scale metabolite generation and purification and circumvents technical complications associated with online LC-NMR. Most importantly, the turnaround time of metabolite structure determination for metabolically unstable compounds using the present methodology is more in sync with the cycle time during which medicinal chemists modify metabolic softspots while performing other iterative lead optimisation activities, demonstrating a real impact on the drug-discovery process.