Stimulated emission depletion (STED) microscopy is a powerful super-resolution imaging technique that only recently entered the field of mitosis, where it proved to be invaluable for studying various microtubule classes, kinetochore-microtubule attachments and chromosome segregation errors. Here, we describe immunofluorescence combined with STED microscopy as a method for analyzing microtubules and kinetochore-microtubule attachments in human mitotic spindles. We also describe live-cell STED microscopy as a method for single-plane short-term imaging of transient processes in crowded spindle areas. Finally, we outline image analysis approaches for the quantitative assessment of microtubule bundles within the spindle.