This review describes a new method for quantitative measurement and spatial imaging of microviscosity within individual domains of live cells. The method is based on fluorescence detection from small synthetic molecules termed 'molecular rotors', which are characterised by a strong response of fluorescence lifetimes or spectra to the viscosity of their immediate environment. We have demonstrated that the quantitative determination of viscosity is possible using lifetime-based molecular rotors and ratiometric molecular rotors. The ratiometric imaging of viscosity benefits from a very fast signal acquisition. We have illustrated this advantage by monitoring changes in intracellular viscosity during photodynamic therapy, which is a clinically utilised modality for the treatment of neoplastic disease.