University students are at higher risk of using psychoactive substances (PS). The aim of this study was to provide insight into the extent and the pattern of PS use among students in four main universities in the West Bank, Palestine. The most frequent lifetime substance uses were smoking tobacco (41.6% for cigarettes and 50% for hookah), drinking alcohol (5.4%), and using illicit drugs (3.0%). The frequency of use was higher among males, with higher income, orphans, or whose parents experienced more conflict in their relationship. Cannabis, synthetic cannabinoids, and amphetamines were the most common used illicit drugs. Factors determined the danger of using PS were the initiation age, availability of money and substances, substances types, frequency of use, and using multiple substances at the same time. Escaping from problems and peer pressure was the most important motives to substances use. Students who manifested a history of violence were significantly more likely being substance users whatever is the substance in question. Overall, PS use is common among Palestinian university students despite them being knowledgeable about the dangers of substance use. The results of this study demonstrate an urgent need for the development of intervention and immediate attention of local authorities.