Focal axonal beading and focal disruption of microtubule structure are characteristic to traumatic axonal injury. We have recently reproduced these morphological and structural changes in our in vitro model system [D. Kilinc, G. Gallo, K.A. Barbee, Mechanically induced membrane poration causes axonal beading and localized cytoskeletal damage, Exp. Neurol. 212 (2008) 422–430]. In order to measure bead formation objectively, an observer-independent quantification of beading was necessary. In addition, a quantitative measure for the extent of co-localization of axonal beads and microtubule disruptions was required to establish a causal relationship between focal cytoskeletal damage and bead formation. In this paper we describe Matlab-based, interactive image analysis programs for axonal beading quantification and co-localization analysis. Injury-induced increases in the axonal beading could be successfully detected using the bead analysis program.