PTW riders are considered to be vulnerable road users as they exhibit high accident and severity rates. There is a slight reduction trend of PTW accidents in Europe over the last years; however this reduction is considerably lower than the corresponding one for car users. At the same time, PTW ownership and use is rising for several reasons including low purchase and use costs, greater movement and parking flexibility in the congested networks, increased popularity of PTW riding as a leisure activity and increasing number of returning riders. Hence, the design of suitable countermeasures that can mitigate PTW accidents is of crucial importance. The aim of this paper is to determine and test a methodological framework to assess road safety measures for PTWs as well as the assessment of existing road safety measures with the use of the proposed methodology. In addition, specific domains which ask for the design and implementation of countermeasures taking into account several effectiveness attributes including the need for such measures, their anticipated impact, acceptance and sustainability as well as the rider perspective are also identified. Research priorities and methodological recommendations are then discussed.