The complexity of biology requires that measurements of biomolecular interactions be performed inside living cells. While electrophoresis inside cells is prohibited by the cell membrane, the movement of molecules along a temperature gradient appears feasible. This thermophoresis could be used to quantify binding affinities in vitro at picomolar levels and perform pharmaceutical fragment screens. Here we changed the measurement paradigm to enable measurements inside living cells. The temperature gradient is now applied along the optical axis and measures thermophoretic properties for each pixel of the camera image. We verify the approach for polystyrene beads and DNA of various lengths using finite element modeling. Thermophoresis inside living cells is able to record thermophoretic mobilities and intracellular diffusion coefficients across the whole cytoplasm. Interestingly, we find a 30-fold reduced diffusion coefficient inside the cell, indicating that molecular movement across the cell cytoplasm is slowed down due to molecular crowding.