The human chorionic gonadotropin (hCG) proteins constitute a diverse group of molecules that displays biomarker value in pregnancy detection and cancer diagnostics, as well as in doping analysis. For the quantification of hCGβ and qualitative differentiation between other hCG variants in a selective, sensitive, and reproducible manner, the targeted proteomics approach based on mass spectrometric (MS) selected reaction monitoring (SRM) detection was exploited. By optimizing immunoaffinity extraction using monoclonal antibodies coated to magnetic beads, access was granted for the MS to the low-abundance target proteins, ensuring proper sensitivity with limits of detection (LODs) of 2 and 5 IU/L, respectively, for urine and serum samples. Validation according to key elements and recommendations defined by the European Medicines Agency in Guideline on Validation of Bioanalytical Methods was performed. For both matrixes this demonstrated good within-day precision results (within 20% for the lowest concentration, and within 15% for the medium and high concentration), good accuracy results (within 15% for all concentrations), and proper linearity, >0.997 for serum and of 0.999 for urine, in the concentration range up to 5000 IU/L. The method's application in clinical diagnostics was tested on samples from a pregnant woman and from patients previously diagnosed with testicular cancer. For doping analysis, samples from one man having received injection of the hCG-containing pharmaceutical Pregnyl were analyzed. The method proved to be quantitatively accurate with indisputable identification specificity, reducing risks of false positive and false negative results. The successfully validated method advocates thus for more extended use of MS in routine analysis.