CPPs have for numerous years been utilized as delivery vectors of various pharmaceutically interesting cargoes, both in vitro and in vivo. As CPPs are gradually approaching the bedsides, investigating toxicity associated with these highly interesting peptides becomes increasingly important and thorough initial assessment of cytotoxicity in vitro is a first step towards advancing these delivery vehicles in to the clinics. The present chapter describes protocols for four cytotoxicity assays in order to provide a toolbox for toxicity assessment of CPPs. The foci lie on membrane integrity (deoxyglucose leakage and propidium iodide assays) and cell viability (the MTT assay), but the chapter also provides a protocol for assessing an important parameter for future clinical applications, namely the hemolytic properties of CPPs.