Transcription kinetics of actively transcribing genes in vivo have generally been measured using tandem gene arrays. However, tandem arrays do not reflect the endogenous state of genome organization in which genes appear as single alleles. Here we present a robust technique for the quantification of mRNA synthesis from a single allele in real time in single living mammalian cells. The protocol describes how to generate cell clones harboring an MS2-tagged allele and how to detect in vivo transcription from this tagged allele at high spatial and temporal resolution throughout the cell cycle. Quantification of nascent mRNAs produced from the single tagged allele is performed using RNA fluorescence in situ hybridization (FISH) and live-cell imaging. Subsequent analyses and data modeling detailed in the protocol include measurements of transcription rates of RNA polymerase II, determination of the number of polymerases recruited to the tagged allele and measurement of the spacing between polymerases. Generation of the cells containing the single tagged alleles should take up to 1 month; RNA FISH or live-cell imaging will require an additional week.