The potent and pluripotent cytokine TGFβ has important roles in normal homeostasis and disease pathogenesis. Once released from cells, TGFβ exists in both latent and functionally active forms. Large amounts of latent TGFβ are secreted from cells and sequestered in extracellular matrix, only a small proportion of which is activated at any given time. Accurate assessment of TGFβ activity levels is an important measurement in biological research and requires methods distinct from measuring total levels of TGFβ expression as small changes in TGFβ activity levels could be masked by the large amounts of latent TGFβ available to be measured. In this chapter, we describe detailed experimental methods for assessing levels of active TGFβ in cells and tissues. This chapter includes methods for the assessment of TGFβ activity in cells in vitro, in ex vivo precision cut tissue, and in vivo.