The diagnostic immunology laboratory relies heavily on protein measurements, especially with the explosion of clinically relevant biomarker analysis. Of particular import are the tremendous advances that have been made in the technology for protein detection, and while not all of it has gained traction in the clinical immunology laboratory, this remains an area of huge growth. However, regulatory processes have not kept up with the burgeoning research in the area of protein analysis, and new diagnostic tests for protein analytes are approved for clinical testing at a glacial pace. Nonetheless, it is critical for the clinical immunologist to understand these advances and determine how they can best be utilized in the clinical laboratory. Besides keeping pace with the rapidly changing technology, the age-old fundamental principles of analytical validation of new tests, protein based or not, are still applicable. This chapter covers the basic principles of protein testing in the clinical laboratory and provides special emphasis on the role of mass spectrometry (MS) in diagnostic protein analysis.