This unit provides the protocols necessary for the quantification of mouse single joint T cell receptor excision circles (sjTRECs) generated during TCRA gene rearrangement. These nonreplicated episomal circles of DNA are generated by the recombination process used to produce antigen‐specific T cell receptors. The number of sjTRECs per mg of thymus tissue or per 100,000 lysed cells has been shown to be a molecular marker of thymopoiesis and naïve T cells. This technology is beneficial to investigators interested in quantitating the level of thymopoiesis occurring in both in vivo and in vitro mouse systems and complements traditional phenotypic analyses of thymopoiesis. The for real‐time PCR details the assay for use on a Bio‐Rad iCycler iQ. An is provided for an ABI Prism 7700. In addition, the unit includes Support Protocols for preparation of a mouse sjTREC DNA standard () and preparation of mouse thymus tissue DNA () as well as a protocol for proteinase K lysis of lymphocytes () used in the Basic and Alternate protocols.