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Secretory proteins play important roles in the cross-talk of individual functional units, including cells. Since secretory proteins are essential for signal transduction, they are closely related with disease development, including metabolic and neural diseases. In metabolic diseases, adipokines, myokines, and hepatokines are secreted from respective organs under specific environmental conditions, and play roles in glucose homeostasis, angiogenesis, and inflammation. In neural diseases, astrocytes and microglia cells secrete cytokines and chemokines that play roles in neurotoxic and neuroprotective responses. Mass spectrometry-based secretome profiling is a powerful strategy to identify and characterize secretory proteins. This strategy involves stepwise processes such as the collection of conditioned medium (CM) containing secretome proteins and concentration of the CM, peptide preparation, mass analysis, database search, and filtering of secretory proteins; each step requires certain conditions to obtain reliable results. Proteomic analysis of extracellular vesicles has become a new research focus for understanding the additional extracellular functions of intracellular proteins. Here, we provide a review of the insights obtained from secretome analyses with regard to disease mechanisms, and highlight the future prospects of this technology. Continued research in this field is expected to provide valuable information on cell-to-cell communication and uncover new pathological mechanisms.