(A) Typical TIRF-based assay to reconstitute and measure microtubule dynamics in vitro in which a GMPCPP-stabilized microtubule seed is immobilized on a passivated glass surface that nucleates a microtubule. The seed and free tubulin typically contain 5–20% tubulin that is labelled with different fluorophores. Microtubule-binding proteins or small molecules can be added and their binding as well as effect on microtubule dynamics measured in this assay. (B) Montage and life history plot of a dynamic microtubule with seed (red), dynamic microtubule lattice (blue) and microtubule tip labelled with EB3-GFP (green). Phases are labelled as growth and shrinkage, transitions marked as catastrophe and rescue. (C) Workflow to measure microtubule dynamics parameters including the generation of kymographs (space–time plots), automatic or manual detection of the edge and extraction of speed and transition rates. The final violin plots are vertical histograms that show the full distribution of growth and shrinkage speeds that make up the median, which is also depicted by an inset boxplot.