The optimisation of flow cytometers is a critical pre-requisite to the generation of high-quality flow cytometry data, as well as tracking instrument performance over time. The determination of appropriate photomultiplier (PMT) voltages in particular is crucial in collecting quality data, especially of high-end and low-end signals, as well as setting proper compensation. The standard instrument setup using BD’s CS&T program ensures proper instrument functionality, but sets PMT voltages to levels that are not practical for many experiments. A number of other approaches have been developed using cells or beads in an attempt to generate optimal cytometer settings, but each is limited as they are often panel-specific, inflexible to changes in the experimental setup, or are non-automated and therefore may not be performed daily. Here we describe a novel optimisation program, which combines the use of the CS&T program, unstained cells, and a number of fluorescent beads to characterize instrument performance. This program determines PMT voltage values for each detector that establish a linear working range where low-end signal is detectible above background and electronic noise, and high-end signal of specific fluorophores is maintained within the linear range of the detector (on-scale). This high-end signal limit is applicable for the vast majority of antigens that are highly expressed on mouse cells, including MHCII, CD11b, and Ly6C. These values are then captured using the BD DIVA application setting, and voltages are set automatically each day by the BD DIVA Application Settings via the CS&T program to achieve these pre-optimised settings. The performance of the cytometer can then be tracked using the standard CS&T program, and experiments run weeks apart can be compared directly. In summary, we have developed a novel program for cytometer optimisation that resulted in automated generation of pre-optimised cytometer settings leading to the generation of high-quality flow cytometry data.