Single-particle tracking (SPT) applications have been growing rapidly in the field of cell biology, and in particular in neurobiology, as a means of unravelling the involvement of diffusion dynamics of neurotransmitter receptors and other synaptic proteins in the regulation of neuronal activity. Suitable probes and technological improvements make SPT more accessible than it used to be and open up broad applications in cellular biology. In this technical highlight, we give an overview of the experimental approach in SPT. The concepts and results in neurobiology have already been the object of detailed reviews. Here, we focus on a qualitative description of the implementation of SPT, from molecule labelling to acquisition, data treatment and analysis of protein diffusion properties. Constraints, limitations and future developments are discussed.