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Abstract. A graphical user interface was designed to facilitate field application of premixing in-line injection sprayers. The primary functions of the interface were: 1) detecting individual components separately before the sprayer operation to make sure they were functional; 2) setting mixture concentrations with resolution of 0.1% in real-time; 3) using virtual level gauge widget to indicate the liquid level in premixing, buffer chemical and water tanks, to help operators know the working process of this system; 4) preventing tanks from overflow; 5) rinsing premixing and buffer tanks, chemical line, and spray nozzles automatically after spray applications. Moreover, this newly designed interface was incorporated with the premixing in-line injection system, and was attached to a variable-rate sprayer tested in field. To assess the chemical concentration accuracy and mixture uniformity, artificial targets were mounted on tree branches to collect spray samples. Viscosity modified fluid was mixed with fluorescent tracer then used as simulated pesticide to validate accuracy of measured spray mixture concentrations. Field experiments demonstrated that the newly designed graphical user interface was user friendly, easy to understand and efficient to operate for operators. In addition, measured concentrations of the spray mixture agreed well with the target concentrations. At target concentrations of 1.0%, the overall relative error (RE) was 7.15% and the coefficient of variation (CV) was 2.85%. Similarly, at target concentration of 2.0%, the overall RE was 4.38%; the CV was 4.35%, respectively. Thus, the premixing in-line injection system was able to provide spray mixtures with acceptable chemical concentration accuracy and desirable mixture uniformity for variable-rate spray applications.