1.1 Prologue Autophagy (“self-eating”) is a highly conserved intracellular degradation process. Recently, an astonishing number of connections to human physiology and disease for autophagy have been made. Thus, autophagy plays a critical role during development and differentiation, in cancer, neurodegeneration, combating viral and microbial infections, and has been linked to life span extension and ageing. For these reasons, the ability to follow autophagy in living mammalian cells is of particular interest, both in terms of developing better understanding at a mechanistic level and in terms of possible future clinical applications. In this chapter our intention is not to summarise the available autophagy assays, but to highlight the application of biosensors used to monitor autophagic processes in live cells. Firstly, we briefly outline current knowledge about the molecular mechanism and function of autophagy (Section 1.2). Then, we outline how autophagy can be measured in live cells in a non-invasive manner and indicate some of the advantages and disadvantages of the biosensor-based assays of autophagy currently in use (Section 2). In particular we focus here on the design and use of Rosella a dual wavelength emission biosensor based on a fusion of fluorescent proteins whose use exploits alterations in pH during autophagy. The advantages of this approach will be highlighted (Section 2.3) and illustrated by applications to follow autophagy both in yeast, an important eukaryotic model organism (Section 2.3.1) and mammalian cells (Section 2.3.2). Finally, we address the future prospects for application of alternative approaches (Section 3) to the measurement of autophagy based on the use of novel probes.