Membrane traffic pathways play an essential role in cells, providing a mechanism for organelles of the endomembrane system to communicate and exchange material between each other. A significant number of infections and diseases are associated with trafficking pathways, and as such gaining a greater understanding of their regulation is essential. Fluorescence-based imaging techniques are widely used to probe the trafficking machinery within cells, and many of these methods have the potential to be applied in a quantitative manner. In the present mini-review, we highlight several recent examples of how image intensity, kinetic measurements, co-localization and texture feature analysis have been used to study the function of one key family of membrane traffic regulators, the Rab GTPases. We give specific emphasis to the importance of the quantitative nature of these recent studies and comment on their potential applicability to a high-throughput format.