This study examined the perceptions of lecturers and students regarding portfolio assessment practices, their overall attitudes towards this assessment method, and the alignment and divergence between their perceptions. Employing a survey research design, the study was conducted at the University of Education, Winneba, in the Central Region of Ghana, involving 52 final-year science education students and five of their lecturers. Students were selected using stratified random sampling, while lecturers were chosen purposefully. Data collection instruments comprised five-point Likert scale questionnaires administered to both groups, ranging from strongly disagree to strongly agree. Descriptive analysis, including mean, frequencies, and percentages, was utilised for data analysis. Results indicated that lecturers generally perceive a clear purpose for portfolio assessment practices (mean score = 4.20) and emphasise integrating them into every educational programme (mean score = 4.40), while students express lower levels of satisfaction with these aspects (mean scores = 2.15, 2.42, respectively). Additionally, lecturers exhibit moderate attitudes towards portfolio assessment (60.0%), while students predominantly demonstrate low attitudes (59.6%). Alignment between lecturers and students is evident in some areas, such as the importance of communicating objectives and providing feedback to students and the importance of this assessment in diagnosing students’ strengths and weaknesses, but discrepancies arise regarding the guidance of students during the practices. The study concluded that understanding and addressing the differences in perceptions and attitudes between lecturers and students is crucial for promoting a shared understanding and acceptance of portfolio assessment practices. In order to close gaps and foster meaningful engagement, it is then recommended that institutions prioritise efforts to improve communication, collaboration, and support for both lecturers and students to enhance the effectiveness of portfolio assessment practices in higher education.