During the past few years, significant technical effort was made to develop molecular methods for the absolute quantitation of nucleic acids in biological samples. In virology, semi-quantitative and quantitative techniques of different principle, complexity, and reliability were designed, optimized, and applied in basic and clinical researches. The principal data obtained in successful pilot applications in vivo are reported in this paper and show the real usefulness of these methods to understand more details of the natural history of viral diseases and to monitor specific anti-viral treatments in real time. Theoretical considerations and practical applications indicate that the competitive polymerase chain reaction (cPCR) and competitive reverse-transcription PCR (cRT-PCR) assay systems share several advantages over other quantitative molecular methodologies, thus suggesting that these techniques are the methods of choice for the absolute quantitation of viral nucleic acids present in low amounts in biological samples. Although minor obstacles to a wide use of these quantitative methods in clinical virology still remain, further technical evolution is possible, thus making the quantitative procedures easier and apt to routine applications.