The present study examined the effects of domestic cooking methods on the biotin contents of Korean foods. Immunoaffinity column cleanup extraction and high-performance liquid chromatography were used to determine biotin contents. Linearity, accuracy, and limits of detection and quantification were acceptable based on the AOAC guidelines. Among legumes, raw soybeans had the highest biotin level, but boiling legumes significantly reduced biotin contents. In contrast, roasting increased biotin contents, but blanching and steaming reduced the biotin contents of most vegetables compared to uncooked vegetables. The study provides reliable data on the biotin contents of Korean agricultural foods.