Multiparameter flow cytometry is an important tool for biomedical scientists, especially for preclinical drug and vaccine research. It is an invaluable tool for detailed analysis of receptors, signaling, and effector molecules as well as nucleic acids in individual cells and cell populations. Technological advancements have made it possible to design and execute flow cytometry studies that simultaneously evaluate multiple parameters. However, evaluating multiple parameters at the cellular level is not an easy task. Below are some sample preparation, panel design, and data analysis aspects for new and inexperienced flow cytometry users to consider.