As cities across North America install infrastructure to accommodate a growing number and variety of bicyclists, installation of bicycle-specific traffic signals is a common design element. A recent survey showed a lack of consistency in design and timing. In particular, minimum green signal timing is highly dependent on the assumed acceleration and speed performance of bicyclists, but no detailed methodology exists to estimate these performance values. However, recent American Association of State Highway and Transportation Officials (AASHTO), Caltrans and National Association of City Transportation Officials (NACTO) documents require that an adequate clearance interval shall be provided, and that in determining this minimum interval field investigation of bicyclists’ speeds is recommended. Furthermore, even if detailed video trajectories are available, the determination of a value for field speed and acceleration is not trivial because values of speeds and accelerations are a function of time and individual bicyclist performance. The purpose of this research is to develop and apply a general methodology to estimate bicyclists’ acceleration and speed for traffic signal timing applications. Utilizing physical equations of motion, this research analytically derives expressions that can be used to classify an individual bicyclist’s performance as a function of the observed acceleration profile. The analysis indicates that four basic acceleration profiles are possible and the profiles can be obtained using a parsimonious field-data collection method. The methodology is successfully applied to two intersections in Portland, OR. A detailed statistical analysis shows that the results are intuitive and that the methodology successfully categorizes bicyclists’ performance variations due to topography or demographic characteristics.