Human chorionic gonadotropin (hCG) is a 237 aminoacid glycoprotein hormone composed of two dissimilar α and β subunits noncovalently linked by charge interactions, which are both required for the biological activity of the hormone. Due to structural heterogeneity, hCG exists in biological fluids as a mixture of different isoforms, i.e., intact active hormone (hCG), nicked hCG (hCGn), free β subunits (hCGβ), free α subunit (hCGα), β-core fragment (hCGβcf, predominantly detected in urine and containing amino acids 6-40 and 55-92 linked by disulphide bridges) and nicked free β-subunit (hCGβn). Although the measurement of hCG might be useful in a kaleidoscope of clinical conditions, such as diagnosis, monitoring and follow-up of pregnancy-related disorders, prenatal screening and gynecological cancers, the leading application is still the diagnosis of pregnancy, where it can be measured quantitatively either in serum or urine, in the latter case also using qualitative and rapid immunoassays. Since there is still debate as to whether serum or urine tests are to be preferred for establishing a diagnosis of pregnancy, we discuss here the main analytical and clinical aspects of hCG measurement for the diagnosis of pregnancy, highlighting the advantages and limitations of assessing hCG in urine and serum.