The widespread adoption of robotic technologies in healthcare has opened up new perspectives for enhancing accuracy, effectiveness and quality of medical procedures and patients’ care. Special attention has been given to the reliability of robots when operating in environments shared with humans and to the users’ safety, especially in case of mobile platforms able to navigate autonomously. From the analysis of the literature, it emerges that navigation tests carried out in a hospital environment are preliminary and not standardized. This paper aims to overcome the limitations in the assessment of autonomous mobile robots navigating in hospital environments by proposing: (i) a structured benchmarking protocol composed of a set of standardized tests, taking into account conditions with increasing complexity, (ii) a set of quantitative performance metrics. The proposed approach has been used in a realistic setting to assess the performance of two robotic platforms, namely HOSBOT and TIAGo, with different technical features and developed for different applications in a clinical scenario.