The immune system consists of a complex network of immune cell subpopulations, with a wide range of cellular markers indicative of cellular identify, activation status, migrational status, and functional status. Moreover, the expansion of fluorescence flow cytometry technology, in conjunction with our ever-expanding understanding of the complexity of the immune system, has lead to the generation of larger polychromatic flow cytometry (PFC) panels. However, the generation of these panels is often difficult and cumbersome, can result in poor quality data if done incorrectly. Here we describe an extremely detailed and yet simple, intuitive process for designing high-quality PFC panels of up to 18 colours, including: hardware (cytometer) configuration and fluorophore options, matching antigen density to fluorophore brightness, dealing with fluorescence spillover and spreading error, and getting the right combinations. Whilst the process generates high quality panels, many practical considerations limit the ability for users to adhere to such a process. Therefore, we have described solutions to numerous practical considerations in the process of panel design including reagent costs, titrations, commercial availability, and laboratory-specific “skeleton” starting panels. Additionally we describe and review the performance and integration of the novel Brilliant Violet and Brilliant Ultra Violet dyes into PFC panels. Finally, we will show practical examples of 16-colour panel generation from our laboratory, as well as specialised approaches on a 10-laser cytometry system.