The leaf hairs (trichomes) on the aerial surface of many plant species play important roles in phytochemical production and herbivore protection, and have significant applications in the chemical and agricultural industries. Trichome formation in the model plant Arabidopsis thaliana also presents a tractable experimental system to study cell differentiation and pattern formation in plants and animals. Studies of this developmental process suggest that trichome positioning may be the result of a self-forming pattern, emerging from a lateral inhibition mechanism determined by a network of regulatory factors. Critical to the continued success of these studies is the ability to quantitatively characterize trichome pattern phenotypes in response to mutations in the genes that regulate this process. Advanced protocols for the observation of changes in trichome patterns can be expensive and/or time consuming, and lack user-friendly analysis tools. In order to address some of these challenges, we describe here a strategy based on polarized light microscopy for the quick and accurate measurement of trichome positions, and provide an online tool designed for the quantitative analyses of trichome number, density and patterning.