Road irregularities generate vibrations that are transmitted to cyclists’ hands. This paper describes a purpose-designed laboratory setup and data processing method to assess vibration-induced numbness and discomfort. The rear wheel of a road bike was coupled with a smart trainer for indoor cycling, while the front wheel was supported by a vibrating platform to simulate road–bike interaction. The vibrotactile perception threshold (VPT) is measured in the fingers, and a questionnaire was used to assess the discomfort in different parts of the hand using a unipolar scale. To validate the method, ten male volunteers underwent two one-hour cycling sessions, one for each of the two handlebar designs tested. VPT was measured in the index and little fingers of the right hand at 8 and 31.5 Hz before and after each session, while the discomfort questionnaire was completed at the end of each session. The discomfort scores showed a strong inter-subject variability, indicating the necessity to combine them with the objective measurements of the VPT, which is shown to be sensitive in identifying the perception shift due to vibration exposure and the differences between the fingers. This study demonstrates the effectiveness of the proposed method for assessing hand numbness and discomfort in cyclists.