All new vehicle types within the European Union must now be equipped with a driver drowsiness and attention warning system starting from 2022. The specific requirements for the test procedure necessary for type approval are defined in the Annex of EU Regulation C/2021/2639. The objectives of this study were to: (i) investigate how sleepiness develops in professional truck drivers under real-road driving conditions; and (ii) assess the feasibility of a test procedure for validating driver drowsiness and attention warning systems according to the EU regulation. Twenty-four professional truck drivers participated in the test. They drove for 180 km on a dual-lane motorway, first during daytime after a normal night's sleep and then at nighttime after being awake since early morning. The results showed higher sleepiness levels during nighttime driving compared with daytime, with a faster increase in sleepiness with distance driven, especially during the night. Psychomotor vigilance task results corroborated these findings. From a driver drowsiness and attention warning testing perspective, the study design with sleep-deprived drivers at night was successful in inducing the targeted sleepiness level of a Karolinska Sleepiness Scale score of ≥ 8. Many drivers who reported a Karolinska Sleepiness Scale ≥ 8 during the drives also acknowledged feeling sleepy in the post-drive questionnaire. Reaching high levels of sleepiness on real roads during daytime is more problematic, not the least from legal and ethical perspectives as higher traffic densities during the daytime lead to increased risks.