Background Two-wheeled vehicles are the major mode of transportation in India. Such vehicles are exposed to excessive vibration on the road when compared to four-wheeled vehicles. However, the research on the reduction of whole body vibration in the case of two-wheelers is not explored in detail. The present study predicts rider comfort in the case of an electric two-wheeler as per ISO 2631-1, by obtaining the finding the weighted acceleration at the strategic locations of vibration at the test rig. Methods An electric two-wheeler test rig is used in the study. The values of acceleration from the test rig in running conditions are obtained by using NI LabVIEW 2019. The drive cycle of the electric vehicle (EV) test rig is controlled by Sync sols’ EV lab software. Obtaining the weighted root mean square (RMS) acceleration from running the test setup, it is compared with the ISO 2631-1 standard to obtain the rider comfort. Results Loading area, traction motor, base mount, and suspension were found to be the strategic points of vibration. Frequency weighted RMS acceleration of 0.3 to 0.4 m/s2 obtained at these points are prone to cause discomfort for the rider. Vehicle speed, road profile, and duration of exposure were found to be important parameters affecting the rider’s comfort. A maximum of 4.6 m/s2 amplitude was observed. The loading area, which corresponds to a rider’s seat in actual vehicle, is important and reduction of these vibrations make the ride comfortable for the rider. Suspension and base mount of the test rig are found to be uncomfortable observing the weighted RMS acceleration. Conclusions A suitable damping technique design is very much essential in reducing these vibrations and improve the rider comfort, as many more non-deterministic vibrations are prone to cause dis-comfort in case of actual on road riding conditions.