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The transforming growth factor-β (TGF-β) is a multifunctional cytokine critical for embryogenesis and tissue homeostasis. Alterations in TGF-β signaling pathway are observed in several types of malignant tumors and often related with cancer progression and metastasis. TGF-β signaling is transduced across the plasma membrane after ligand-receptor binding and consequent phosphorylation of the intracellular effectors SMAD2/3 by TGF-β receptors. Phosphorylated SMAD2/3 accumulates in the nucleus after complex formation with SMAD4 to act as transcription factors and regulate the expression of genes critically associated with cell proliferation and differentiation. Traditional methodologies used to assess TGF-β signaling pathway lack accuracy and/or show poor scalability, limiting in vitro experiments and almost excluding their use in vivo. Here, we describe a fast method to quantitate TGF-β signaling pathway activity in vitro and in vivo by using adenoviral reporters. Its implementation in vitro allows quantitating cell response to TGF-β at concentrations as low as pictograms/mL. Additionally, the use of an in vivo imaging system (IVIS) enables quantitating and monitoring TGF-β signaling pathway activity over time during cancer progression, eliminating the requirement of animal euthanasia at multiple time points for this purpose. Importantly, this protocol has been consistently used in different models and effectively led to the visualization and measurement of TGF-β activity levels. Improving the sensitivity, specificity, and scalability of methods focused on characterizing this and other molecular pathways will result in a better understanding of their biology in physiological and pathological processes.