Bacterial chemotaxis is an important behavior to study to understand the spatial segregation of species in mixed communities and the assembly of host microbiomes. This is particularly relevant in the rhizosphere, where chemoattraction towards root exudates is an important determinant of plant colonization. However, current methods to screen chemoeffectors are limited in their throughput, creating a barrier to generating comprehensive datasets describing chemotactic profiles for species of interest. Here, we describe a novel, 3D-printed capillary tube holder approach, which enables up to 384 simultaneous capillary tube chemotaxis assays. We optimized and benchmarked our assay using Escherichia coli k12 and Bacillus subtilis 3610 and their known chemoattractants: serine and aspartate. We then tested the threshold concentration of these chemoattractants in our assay and found we could detect chemoattraction towards concentrations spanning multiple orders of magnitude. In this paper, we describe our novel high-throughput chemotaxis assay in detail and provide the necessary files for 3D printing the capillary tube holder.