Metanephrines (metanephrine [MN] and normetanephrine [NMN]) are O-methylated metabolites derived from the catecholamines, epinephrine, and norepinephrine, respectively. High concentrations of metanephrines have been observed in individuals with pheochromocytoma, a neuroendocrine tumor. Measurement of metanephrines in urine is used to screen for the tumor. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended due to the high sensitivity, specificity, and throughput of the technique. Herein, we describe an optimized LC-MS/MS assay for the analysis of urinary metanephrines.