Interest in the use of neurophysiological instruments for real-world studies in the workplace is increasing, also intensified by the simultaneously growing use of various commercial self-tracking technologies. However, the application of neurophysiological tools for real-world workplace research is associated with challenges—an aspect that has received little attention in previous research. This article outlines the key challenges encountered when applying neurophysiological measurements in the workplace, drawing on insights gained in an interdisciplinary research project on digital workplaces. We identify challenges along four main themes: technical tool requirements, data processing and interpretation, tool interaction, and organizational collaboration. Additionally, we discuss how these challenges were addressed within our case. As a contribution, this article offers important considerations and recommendations for the effective application of neurophysiological tools in real-world workplace research.