<p>Entrepreneurial Mindset (EM) has emerged as a key driver of innovation, employability, and societal impact within Higher Education Institutions (HEIs), underpinning their evolving “third mission” beyond teaching and research. Yet, despite its growing prominence, EM in higher education lacks integrated, empirically tested measurement tools. This study designs and pilots a multilevel diagnostic instrument capturing EM across individual, institutional, and ecosystem levels. Informed by a systematic review and expert input, the instrument was piloted at a Greek university. High reliability of the items indicated sound construct validity. Twelve theory-driven hypotheses were evaluated using Spearman rank coefficient to identify correlations amongst them. The associations demonstrated different scales of correlation for hypotheses enabling culture and institutional supports align with intrapreneurial behaviours, structured external engagement connects to ecosystem linkages, and experiential learning relates to leadership and venture intentions. The strongest links connected faculty EM with an established culture of academic intrapreneurship and, more broadly, institutional supports with external collaboration. Weaker but meaningful links tied funding mechanisms to start-up/spinoff outcomes and experiential learning curriculum to leadership skills. Results substantiate EM as a tightly coupled, multilevel construct in HEIs and position the instrument as a practical diagnostic for strategy and benchmarking. Beyond measurement, this instrument can function as a practical tool for HEIs supporting their strategic planning, resource allocation, and benchmarking of their entrepreneurial initiatives. The research, lastly, identifies actionable levers, such as culture change, support infrastructures, and ecosystem engagement, for aligning EM development with universities’ third mission and sustainable and regional transition agendas.</p>