In vitro carrier tests, suspension tests, time-kill curves, and determinations of minimum inhibitory concentrations to evaluate the microbicidal activities of hand antiseptics provide only a preliminary indication of the antimicrobial spectrum and speed of action of a given formulation. Ex vivo testing with human or animal skin at human skin temperature and at contact times reflecting field conditions may give a better indication of a formulation's ability to tackle hand-transmitted pathogens. Field testing of hands for levels of skin microbiota before and after antisepsis may be easier to perform, but it is subject to many uncontrollable factors. Whereas randomised clinical trials may be the ultimate approach to assess the effectiveness of hand hygiene protocols and products in preventing microbial cross-transmission and, ultimately, infections, they can be prohibitively expensive, time-consuming, difficult to design, and therefore impractical. Hence, the primary emphasis should be on in vivo testing on human hands, using a well-designed protocol that closely simulates the recommended field use of the formulation, and possibly followed by clinical studies. The use of these method is the most likely to yield useful data on the potential of a formulation to interrupt the spread of pathogens transmitted by hands in healthcare settings. This review provides a critical assessment of the methods currently used to meet regulatory requirements for hand antiseptics in Europe and North America.