A pillar array column with a perfectly ordered internal structure has a higher separation efficiency than a particle-packed column used in conventional high-performance liquid chromatography. However, applying the pillar array column technology to quantitative analysis is challenging as the injection volume of the sample varies for each injection. This occurs because the sample volumes are in the order of nanoliters and the sample is injected manually. In this study, an automated sample injection system was developed to solve this problem. The system was composed of two pumps (one for the sample and other for the mobile phase), a six-way valve that can be controlled by a PC, and an autosampler. The peak height deviation, which is more than 20% in the conventional manual injection system, was improved to 1.2% and 0.4% for the two coumarin dye samples. This result indicated that the automated sample injection method developed in this study could be applied to pillar array columns to allow for quantitative analysis.