Motivation: Microtubules (MTs) are polarized polymers that are critical for cell structure and axonal transport. They form a bundle in neurons, but beyond that, their organization is relatively unstudied. Results: We present MTQuant, a method for quantifying MT organization using light microscopy, which distills three parameters from MT images: the spacing of MT minus-ends, their average length, and the average number of MTs in a cross-section of the bundle. This method allows for robust and rapid in vivo analysis of MTs, rendering it more practical and more widely applicable than commonly-used electron microscopy reconstructions. MTQuant was successfully validated with three ground truth data sets and applied to over 3000 images of MTs in a C. elegans motor neuron.