The control shifting between a human driver and a semi-autonomous vehicle is one of the most critical scenarios in the road-map of autonomous vehicle development. This paper proposes a methodology to study driver's behaviour in semi-autonomous driving with physiological-sensors-integrated driving simulators. A virtual scenario simulating take-over tasks has been implemented. The behavioural profile of the driver has been defined analysing key metrics collected by the simulator namely lateral position, steering wheel angle, throttle time, brake time, speed, and the take-over time. In addition, heart rate and skin conductance changes have been considered as physiological indicators to assess cognitive workload and reactivity. The methodology has been applied in an experimental study which results are crucial for taking insights on users’ behaviour. Results show that individual different driving styles and performance are able to be distinguished by calculating and elaborating the data collected by the system. This research provides potential directions for establishing a method to characterize a driver's behaviour in a semi-autonomous vehicle.