A nonlinear alignment strategy was examined for the quantitative analysis of serum metabolites. Two small-molecule mixtures with a difference in relative concentration of 20-100% for 10 of the compounds were added to human serum. The metabolomics protocol using UPLC and XCMS for LC-MS data alignment could readily identify 8 of 10 spiked differences among more than 2700 features detected. Normalization of data against a single factor obtained through averaging the XCMS integrated response areas of spiked standards increased the number of identified differences. The original data structure was well preserved using XCMS, but reintegration of identified differences in the original data reduced the number of false positives. Using UPLC for separation resulted in 20% more detected components compared to HPLC. The length of the chromatographic separation also proved to be a crucial parameter for a number of detected features. Moreover, UPLC displayed better retention time reproducibility and signal-to-noise ratios for spiked compounds over HPLC, making this technology more suitable for nontargeted metabolomics applications.