NMR spectroscopy of urine is a fertile bioanalytical approach for a wide range of studies in areas such as toxicity, drug development, molecular epidemiology, disease diagnosis, and nutrition. In this chapter, technical concerns critical to the design and execution of urinary NMR experiments are explored. Beginning with the chemical characteristics of urinary NMR spectra, we discuss the history of urinary NMR metabolomics through studies of toxicity and its suitability as a platform for large-scale studies due to high reproducibility and robustness. With respect to experimental design, a detailed discussion of validated urine collection procedures for both human and other animal model experimental systems is provided along with procedures for the use of preservatives and storage. We explore specific issues in the acquisition of urinary NMR experiments, such as the choice of pulse program and solvent suppression. Data pre-processing techniques, such as spectral binning, quantitative peak-fitting, and full-spectrum approaches, as input to subsequent chemometric evaluation of NMR spectra are detailed. Moving towards applications, we review illustrative biological examples of NMR spectroscopy of urine to studies of normal variation and non-healthy phenotypes. Finally, we discuss emerging challenges in biomarker discovery as well as the emerging field of pharmacometabonomics.