Transforming growth factor-beta (TGF-beta) signalling controls many aspects of cell behaviour and is implicated as a key regulator in tumour formation and progression. However, evaluating levels of active TGF-beta in culture medium or patient plasma and gaining definitive information regarding the activity of downstream substrates such as Sma- and Mad-related protein 3 (Smad3) in vivo with accuracy and sensitivity has been problematic. Therefore, to overcome these technical issues we have created a NIH3T3 cell line with stable pCAGA12-luc expression that can now be utilised to detect TGF-beta activity with high sensitivity. In addition, we have created an adenoviral Smad3 luciferase reporter construct pAd.CAGA12-luc to successfully infect cells for in vitro assays, or prior to injection into mice and used to measure transcriptional activity in vivo. Thus, the NIH3T3-pCAGA12-luc cell line and the pAd.CAGA12-luc adenovirus will be extremely useful tools to measure TGF-beta signalling activity with far greater efficiency and reliability compared to original and currently used reagents.