Here, we describe a detailed protocol, based on laser ablation and fluorescence optical microscopy, to measure the microtubule organization in spindles, including microtubule length distribution, polarity, and plus and minus end densities. The method uses the asymmetry in microtubule depolymerization after a cut, where the newly created microtubule plus ends depolymerize all the way to the minus ends, whereas the newly created minus ends remain stable. The protocol described in this chapter is optimized for spindles, but can be easily applied to any microtubule-based structure. The chapter is divided into two parts. First, we provide the theoretical basis for the method. Second, we describe in detail all steps necessary to reconstruct the microtubule organization of a spindle assembled in Xenopus laevis egg extract. Compared to electron microscopy, which in theory can resolve individual microtubules in spindles andprovide similar structural information, our method is fast and simple enough toallow for a full quantitative reconstruction of the microtubule organizationofseveral X. laevis spindles-which have volumes tens of thousands of timeslargerthan spindles whose structures have been previously solved by electronmicroscopy-in a single experimental session, as well as to explore howthe architecture of these structures changes in response to biochemical perturbations.