Introduction Undergraduate medical research is very important not only for scientific learning but also for career progress. However, there are barriers, especially in developing countries, that restrict undergraduate research. This study aims to evaluate the barriers experienced by medical students in conducting research at undergraduate level. Methods It was an observational, cross-sectional survey conducted with 687 clinical students of two public medical universities of Pakistan. A self-structured questionnaire consisting of seven items was administered to assess the barriers in conducting research at undergraduate level. Data was processed and analysed through SPSS v 22.0 (IBM Corp., Armonk, NY, USA). Results Lack of knowledge as a barrier was identified by 90.68% (n = 623) students. The second most common barrier identified by the students was lack of time (88.79%; n = 610), followed by lack of mentoring as the third most common barrier (85.74%; n = 572). Subgroup analysis showed that lack of knowledge, lack of mentoring, limited data base access, lack of time, and lack of finances were more crucial barriers for female gender (p < 0.05). Only lack of interest was a crucial barrier for male gender (p < 0.05). Conclusion A number of barriers need to be addressed in order to enhance students' participation in clinical research such as lack of interest, funding, and poor availability of research mentors and access to scientific databases to improve participation in clinical research. Substantial amendments in the medical undergraduate curriculum are needed.