**3.4 Summary**

Determination of the exact amounts of proteins gives valuable information about biological mechanisms of targeted proteins and becomes crucial for the evaluation of various biomarker candidates. Until recently, immunoassays such as ELISA were considered as the reference methodology for protein quantification. However, these assays have some critical issues such as difficulty of antibody production and non-specific cross-reactivity of antibodies, and thus a significant investment in time and resources is often required for the accurate evaluation. On this regard, the recent development of MS-based protein quantification techniques using isotope labeled internal standards has become an alternative and compelling approach that provides high accuracy and specificity in protein quantification. Although such techniques are now essential for protein quantification, extensive validation of such procedures is necessary in order to establish metrologically sound analytical techniques.
