Analyzing more than one cellular biomarker (multiplexing) in a single sample is advantageous for a number of reasons. Multiplexing allows researchers to save money and time, while conserving critical samples. In addition, understanding the relationship between cell biomarkers can provide a more complete picture of cell health, leading to improved predictive models for drug discovery. Understanding biomarker relationships can also minimize ambiguity in the data set and validate if a treatment effect is real or an artifact of the system. To avoid repeat experiments and extract the most physiologically relevant data from multiplex cell-based assays, we discuss considerations around assay choices, cell type, cell culture, treatment parameters, detection and appropriate experimental controls.