The genetic integrity of every organism depends on the faithful partitioning of its genome between two daughter cells in mitosis. In all eukaryotes, chromosome segregation requires the assembly of the mitotic spindle, a bipolar array of dynamic microtubules. Perturbations in microtubule dynamics affect spindle assembly and maintenance and ultimately result in aberrant cell divisions. To identify new regulators of microtubule dynamics within the hundreds of mitotic hits, reported in RNAi screens performed in C. elegans, Drosophila and mammalian tissue culture cells [Sonnichsen et al., 2005; Goshima et al., 2007; Neumann et al., 2010], we established a fast and quantitative assay to measure microtubule dynamics in living cells. Here we present a fully automated workflow from RNAi transfection, via image acquisition and data processing, to the quantitative characterization of microtubule behaviour. Candidate genes are knocked down by solid-phase reverse transfection with siRNA oligos in HeLa cells stably expressing EB3-EGFP, a microtubule plus end marker. Mitotic cells are selected using an automatic classifier [Conrad et al., 2011] and imaged on a spinning disk confocal microscope at high temporal and spatial resolution. The time-lapse movies are analysed using a multiple particle tracking software, developed in-house, that automatically detects microtubule plus ends, tracks microtubule growth events over consecutive frames and calculates growth speeds, lengths and lifetimes of the tracked microtubules. The entire assay provides a powerful tool to analyse the effect of essential mitotic genes on microtubule dynamics in living cells and to dissect their contribution in spindle assembly and maintenance.