Myocardin-related transcription factor (MRTF) and the paralogous Hippo pathway effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are transcriptional co-activators that play pivotal roles in myofibroblast generation and activation, and thus the pathogenesis of organ fibrosis. They are regulated by a variety of chemical and mechanical fibrogenic stimuli, primarily at the level of their nucleocytoplasmic shuttling. In this chapter we describe the tools and protocols that allow for exact, quantitative, and automated determination and analysis of the nucleocytoplasmic distribution of endogenous or heterologously expressed MRTF and YAP/TAZ, measured in large cell populations. Dynamic monitoring of nucleocytoplasmic ratios of transcription factors is a novel and important approach, suitable to address both the structural requirements and the regulatory mechanisms underlying transcription factor traffic and the consequent reprogramming of gene expression during fibrogenesis.