The mitotic spindle is a dynamic and complex cellular structure made of microtubules and associated proteins. Although the general localization of most proteins has been identified, the arrangement of the microtubules in the mitotic spindle and precise localization of various proteins are still under intensive research. However, techniques used previously to decipher such puzzles are resolution limited or require complex microscopy systems. On the other hand, expansion microscopy is a novel super-resolution microscopy technique that uses physical expansion of fixed specimens to allow features closer than the diffraction limit of light (~250nm) to become resolvable in the expanded specimen on a conventional confocal microscope. This chapter focuses on expansion microscopy of the mitotic spindle, specifically using tubulin labeling to visualize all microtubule subpopulations within the spindle. Furthermore, we discuss a protocol for expansion of GFP-tagged proteins, such as protein regulator of cytokinesis 1 (PRC1). We also discuss various approaches for image analysis pointing out main advantages of expansion microscopy when compared to previously used techniques. This approach is currently used in our laboratory to study the architecture of the microtubules in the mitotic spindle after perturbations of various proteins important for the structural and dynamical properties of the mitotic spindle.