Accurate flow is important for your chromatography step to provide reproducible results at different scales. For ÄKTA pilot 600 chromatography system, flow and gradient formation is controlled by the UNICORN™ system control software. In this work, flow accuracy was investigated at a combination of different backpressures, flow rates, temperatures, and liquid viscosities. Gradient accuracy and range were calculated from the flow accuracy. Gradient accuracy was also evaluated by running step and linear gradients at different flow rates. Within the buffer B gradient range (%B) from 1%B to 99%B at a system flow rate of 5–600 mL/min, gradient accuracy was found to be ± 1%B.