Immunoreagents formed the basis of early fixed end point high content screening (HCS) assays and their use in HCS applications in drug discovery will continue to increase. One important application of immunoreagents is their incorporation into multiplexed HCS assays in which multiple physiological features are simultaneously measured and related in the same cells. However, creating multiplexed HCS assays that incorporate multiple immunoreagents presents issues such as reagent compatibility, spectral signal overlap, and reproducibility that must be addressed. Here, an example multiplexed fixed end point HCS assay is used to guide potential assay developers on how to optimize complex, yet cellular information rich, multiplexed HCS assays although avoiding some common pitfalls.