We report on the development and characterization of automated metal-free multiple-column nanoLC instrumentation for sensitive and high-throughput analysis of phosphopeptides with mass spectrometry. The system employs a multiple-column capillary LC fluidic design developed for high-throughput analysis of peptides (Anal. Chem. 2001, 73, 3011-3021), incorporating modifications to achieve broad and sensitive analysis of phosphopeptides. The integrated nanoLC columns (50 microm i.d. x 30 cm containing 5 microm C18 particles) and the on-line solid phase extraction columns (150 microm i.d. x 4 cm containing 5 microm C18 particles) were connected to automatic switching valves with non-metal chromatographic accessories, and other modifications to avoid the exposure of the analyte to any metal surfaces during handling, separation, and electrospray ionization. The nanoLC developed provided a separation peak capacity of approximately 250 for phosphopeptides (and approximately 400 for normal peptides). A detection limit of 0.4 fmol was obtained when a linear ion trap tandem mass spectrometer (Finnegan LTQ) was coupled to a 50-microm i.d. column of the nanoLC. The separation power and sensitivity provided by the nanoLC-LTQ enabled identification of approximately 4600 phosphopeptide candidates from approximately 60 microg COS-7 cell tryptic digest followed by IMAC enrichment and approximately 520 tyrosine phosphopeptides from approximately 2mg of human T cells digests followed by phosphotyrosine peptide immunoprecipitation.