Exposure to acute stress can lead to changes in nociceptive thresholds. Laser technique has been used successfully to measure nociceptive thresholds in cattle. This paper presents data from three experiments examining whether: 1) the method is valid as a measurement of nociceptive threshold in dairy cows; 2) there is an acceptable repeatability within 15 min (the duration of most experimental acute stressors); and 3) skin condition (intact vs. naked) affects nociceptive threshold and its variability in dairy cows. Three experiments were performed to observe the behavioural responses to a computer-controlled CO2 laser beam applied to the skin on the caudal aspect of the metatarsus. In experiment 1, effects of power output on nociceptive responses were examined on dairy cows kept and tested in tie stalls. Increasing the power output affected the latencies to respond, types of response, and behaviour during (increasing frequency of tail flicking) and between single laser exposures (increasing frequency of kicking). Therefore, behavioural responses to a laser stimulus seem to be a valid measure of nociception in dairy cows. Repeatability within 15 minutes was investigated in experiment 2. The variables’ latency to move the exposed leg and frequency of tail flicking during laser exposure showed the highest level of repeatability. Effects of power output and skin condition were examined in experiment 3. Increasing the power output and shaving off hair led to increased responses as seen by shorter latencies to respond, less non-responding, and increased kicking. In conclusion the results of these experiments suggest that behavioural responses to laser stimulation are a valid and reliable measure of nociception in dairy cows, especially when applied on naked skin.