The field of metabolomics has been growing tremendously over the recent years and, consistent with that growth, a number of investigators have been looking at the potential of NMR-based urinary metabolomics for several applications. While such applications have shown promising results, there still remains an enormous amount of work to be done before this approach becomes accepted and widely used in clinical diagnostics and other biomedical applications. To achieve such goals, optimization of parameters and standardization of protocols are of paramount importance. In view of this, in this chapter, we present some recommended methods and procedures that can help researchers in the field. Furthermore, we have highlighted some of the challenges encountered in such applications and suggested some possible ways to overcome those challenges.