Phenotypic screen holds great potential in the discovery of new small molecule probes and drugs, as it permits interrogating small molecules with native targets and pathways in model organisms and disease relevant cells. In recent years, label-free cell phenotypic profiling has emerged as an alternative for drug discovery. This paper provides the rationale for phenotypic screens, discusses the basics of label-free cell phenotypic profiling technologies, and provides some guidelines as to how to use these techniques to facilitate drug discovery.