University students are a sub-group of the population at high risk of COVID-19 infection, and their judgments on vaccination affect the public attitudes towards vaccination. Thus, the present study aimed to assess the knowledge, attitudes, perceptions, and acceptance of the COVID-19 vaccine among pharmacy and non-pharmacy students. A cross-sectional study was conducted by enrolling pharmacy (375) and non-pharmacy (225) students from the universities in Lahore. Chi-square analysis was used for significant frequency distributions and a 5-point Likert scale was used to score attitude, perception, and acceptance. The majority of the students were aged between 19–24 years, hailing from urban and middle-class families with good self-reported health. The preferred vaccine was Pfizer, followed by Sinopharm and Sinovac. The major source of information was social media, followed by government campaigns and family members. The pharmacy students demonstrated better knowledge and positive attitudes toward COVID-19 vaccination. The non-pharmacy students scored higher for the questions based on scientific leads, myths, and baffling conspiracies. The non-pharmacy students showed higher hesitancy/barrier total scores related to their trust in the health system, COVID-19 vaccine storage, and efficacy. Data suggested that pharmacy students exhibited better knowledge, positive attitudes, and perceptions about COVID-19 vaccination. Overall, vaccine efficacy and safety were mutual concerns. Nonetheless, non-pharmacy students were hesitant due to mistrust in the health system of Pakistan.