Mass spectrometry (MS)-based proteomic profiling and protein identification has become a powerful tool for the discovery of new disease biomarkers. Among the MS platforms, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) MS offers high sample throughput and the flexibility to couple with different off-line sample fractionation techniques. Here, we present a strategy using MALDI-TOF/TOF MS to analyze fractionated human serum samples for proteomic profiling and then identify serum peptides from these proteomic profiles. We achieve the profiling analyses by using different functionalized magnetic beads to enrich specific subsets of serum proteins/peptides based on their absorption to these beads. This step is followed by elution, transfer onto prestructured sample supports (AnchorChip targets), and analysis in a MALDI-TOF/TOF mass spectrometer. Selected serum peptides are then analyzed in the tandem MS (TOF/TOF) mode to generate fragment ions for determination of their amino acid sequences. We have demonstrated that using this approach, proteomic profiling and protein identification can be done in a single MS instrument.