Primarily, the purpose of hCG detection has been the confirmation of pregnancy by analysis of urine or blood. However, the number of applications for which hCG or hCGβ has been used for diagnostic purposes has significantly grown over the years. Examples of such applications are the detection of hydatidiform mole, cancer, ectopic pregnancy, and fetal trisomies. The wide range of applications renders the accurate and precise detection and quantification of the molecule in various biological fluids necessary. Therefore, in this chapter, the quantification and detection of hCG and its variants using MALDI-ToF mass spectrometry will be discussed.