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There are several advantages to using liquid-filled automated liquid-handling systems equipped with reusable fixed tips for sample handling of bioanalytical assays. However, liquid-handling parameters that have not been optimized can lead to sample dilution by the system liquid of the automated liquid handler causing possible inaccuracy of sample delivery. In this investigation, liquid-handling parameters involving sample delivery, such as aspiration speed, dispense speed, partition volume, excess volume, and air gaps, were closely examined to understand their roles in the accurate delivery of the sample. Consequently, two strategies for optimization of the parameters are presented that achieve accurate sample delivery while maintaining sample integrity.