Metabolite identification and annotation procedures are necessary for the discovery of biomarkers indicative of phenotypes or disease states, but these processes can be bottlenecked by the sheer complexity of biofluids containing thousands of different compounds. Here we describe low-cost novel SPE-NMR protocols utilising different cartridges and conditions, on both natural and artifical urine mixtures, which produce unique retention profiles useful to metabolic profiling. We find that different SPE methods applied to biofluids such as urine can be used to selectively retain metabolites based on compound taxonomy or other key functional groups, reducing peak overlap through concentration and fractionation of unknowns and hence promising greater control over the metabolite annotation/identification process.