The safety effects of cooperative intelligent transport systems (C-ITS) are mostly unknown and associated with uncertainties, because these systems represent emerging technology. This study proposes a bowtie analysis as a conceptual framework for evaluating the safety effect of cooperative intelligent transport systems. These seek to prevent road traffic accidents or mitigate their consequences. Under the assumption of the potential occurrence of a particular single-vehicle accident, three case studies demonstrate the application of the bowtie analysis approach in road traffic safety. The approach utilizes exemplary expert estimates and knowledge from literature on the probability of the occurrence of accident risk factors and of the success of safety measures. Fuzzy set theory is applied to handle uncertainty in expert knowledge. Based on this approach, a useful tool is developed to estimate the effects of safety-related cooperative intelligent transport systems in terms of the expected change in accident occurrence and consequence probability.