As a result of increased sensitivity of molecular methods in detecting microbiological agents, one aspect in the diagnosis of human pathogens has become more and more important: the quantification of infectious agents. In the recent past, real-time PCR-based assays have gained favour for this purpose, since they are characterized by a wide range of quantification (7-8 Logarithmic decades), a high technical sensitivity (< 5 copies) and a high precision (< 2% standard deviation). Time-consuming post-PCR steps are not necessary and have decreased the risk of cross-contamination. The possibility of automated sample preparation has improved turn-around time and allows the development of high throughput screening assays. Introduction of multiplex assays for either subtyping of infectious agents or the simultaneous calculation of pathogen relative to the food stuff has further increased the accuracy of these assays. However, the higher risk of false negative results due to mutations in the primer or probe binding region necessitates to carefully investigate the molecular epidemiology of the pathogen of interest before the design of reliable real-time PCR based assays.