The purpose of this case study is to create a bicycle infrastructure route evaluation method, based on street-level bicycle accessibility. The center of Athens, from Monastiraki to Gyzi, was defined as the evaluation area. Initially, data were collected for each road segment separately in the demarcated study area, from which cyclist's stress levels were extracted. Together with other suitable evaluation criteria, stress levels contributed to the final equation which calculated the “real length” of each route segment based on safety and speed of cyclists and through this procedure the appropriate roads for the Cycling Route Network were chosen. This evaluation method contributes to already known and used methodologies. The methodology allows automatization of bicycle network route choice through a software, or an application.