Many aspects of the physical principles governing protein folding in vitro have been elucidated in the past decades. At the same time, a large number of cellular components involved in protein folding in vivo have been identified. But our mechanistic understanding of how these cellular components affect the energy landscape of the folding process has remained very limited, largely due to a lack of suitable methods. An opportunity to bridge this gap is single molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET), a new technique that allows the investigation of distance distributions and dynamics of individual protein molecules even in complex and heterogeneous environments. In a multi-disciplinary project employing methods ranging from molecular biology and protein biochemistry to optics, microfluidic mixing, and development of instrumentation and software, we plan to use single molecule spectroscopy for investigating the question how proteins find their native three-dimensional structure in a cell. Our initial focus will be on molecular chaperones, which assist protein folding in vivo, but the approach is applicable to a wide range of related phenomena and will be extended to other cellular components. This project is thus expected to nucleate a comprehensive biophysical analysis of intracellular protein folding and dynamics, eventually within live cells. A detailed investigation of these processes will be crucial for understanding the fine balance between protein folding and misfolding in the cell, and the large number of diseases associated with protein misfolding and aggregation.