LED illumination systems for fluorescence microscopy offer a wealth of benefits in comparison to traditional mercury and metal halide lamps, including ease of use, improved stability, and enhanced control. To fully realize these benefits, it is important to ensure that optical filters are configured correctly, which often can be confusing. However, without the correct filter configuration, experimental conditions can be suboptimal, and results may therefore be inaccurate. This article looks at optical filter setup in more depth, explaining the purpose and benefits of optimal LED filtering.