The α-β tubulin heterodimer undergoes subtle conformational changes during microtubule assembly. These can be modulated by external factors, whose effects on microtubule structure can be characterized on 2D views obtained by cryo-electron microscopy. Analysis of microtubule images is facilitated if they are straight enough to interpret and filter their image Fourier transform, which provide useful information concerning the arrangement of tubulin molecules inside the microtubule lattice. Here, we describe the use of the TubuleJ software to straighten microtubules and determine their lattice parameters. Basic 3D reconstructions can be performed to evaluate the relevance of these parameters. This approach can be used to analyze the effects of nucleotide analogues, drugs or MAPs on microtubule structure, or to select microtubule images prior to high-resolution 3D reconstructions.