The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time. [Display omitted] •Live quantification of RhoA activity during development and disease progression•Real-time visualization of RhoA signaling in normal skin, osteocytes, and neutrophils•Monitoring deregulation of RhoA activity in invasive mammary and pancreatic cancers•Longitudinal in vivo imaging of RhoA inhibition using optical windows Nobis et al. generated a RhoA-FRET biosensor mouse to characterize and quantify the spatiotemporal distribution of RhoA activity in native mammalian tissues in vivo during development and disease progression. They show that RhoA activity is tightly regulated during various normal biological processes and is co-opted in disease settings, such as invasive breast and pancreatic cancers.