Parkinson's disease (PD) is an age-associated neurodegenerative disorder hallmarked by a loss of mesencephalic dopaminergic neurons. Accurate recapitulation of the PD movement phenotype in animal models of the disease is critical for understanding disease etiology and developing novel therapeutic treatments. However, most existing behavioral assays currently applied to such animal models fail to adequately detect and subsequently quantify the subtle changes associated with the progressive stages of PD. In this study, we used a video-based analysis system to develop and validate a novel protocol for tracking locomotor performance in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We anticipated that (1) treated mice should use slower, shorter, and less frequent strides and (2) that gait deficits should monotonically increase following MPTP administration, as the effects of neurodegeneration become manifest. Video-based biomechanical analyses, utilizing behavioral measures motivated by the comparative biomechanics literature, were used to quantify gait dynamics over a seven-day period following MPTP treatment. Analyses revealed shuffling behaviors consistent with the gait symptoms of advanced PD in humans. Here we also document dramatic gender-based differences in locomotor performance during the progression of the MPTP-induced lesion, despite male and female mice showing similar losses of striatal dopaminergic cells following MPTP administration. Whereas female mice appeared to be protected against gait deficits, males showed multiple changes in gait kinematics, consistent with the loss of locomotor agility and stability. Overall, these data show that the novel video analysis protocol presented here is a robust method capable of detecting subtle changes in gait biomechanics in a mouse model of PD. Our findings indicate that this method is a useful means by which to easily and economically screen preclinical therapeutic compounds for protecting against or reversing neuropathology associated with PD neurodegeneration.