The e-portfolio is a tool increasingly used in Higher Education due to the benefits it holds, especially with regards to facilitating processes of evaluation and reflective learning. The main goal of this study was the construction of a questionnaire to analyse EP use in a Spanish University and to ensure its reliability and validity for use within relevant samples. To perform this task, we proposed a three-phase procedure: a development phase based on a literature review and the subsequent proposal of items; an evaluation phase, with revision based on experts' judgment; and an analysis phase, in which the instrument's reliability and validity (N = 251) were evaluated through factor analyses and descriptive statistical analyses according to dimension, degree of internal consistency and the relationships uncovered amongst the variables. As a result the Questionnaire on the E-Portfolio in Higher Education is proposed. It is composed of 32 items divided between three dimensions: Pedagogical considerations, academics' teaching and usability. The analyses performed show the questionnaire to be valid and reliable for its proposed purpose. The questionnaire may be useful for HE academics who use the EP and for educational administrators who wish to analyse educational practices of this kind.