Objective: This survey aimed to explore knowledge and experience on novel psychoactive substances (NPS) of healthcare professionals (HCPs). The study also aimed to assess how HCPs would like to improve their knowledge of NPS. Methods: Seventy paper questionnaires were disseminated in 2017 within continuing education events to pharmacists, nurses and general practitioners (GPs). Additionally, 127 online surveys were completed using the Qualtrics platform by other HCPs and mental health nurses in six United Kingdom (UK) independent mental health hospitals long-stay in-patient rehabilitation services. Two educational sessions involving pharmacists and GPs were also held in late 2017 and mid-2018. Knowledge of NPS by HCPs was evaluated prior to the start of the educational events. Evaluation forms were handed out post-sessions to garner feedback, especially on areas for improvement for future sessions. Statistical analysis of data was undertaken using SPSS (V.25). Results: Most HCPs reported only ‘basic’ to ‘intermediate’ NPS knowledge. Substance misuse service staff felt more informed, were more often consulted and had greater confidence regarding NPS compared to hospital and primary care professionals. A negative association was found between the age of the HCP and knowledge of NPS. Most participants expressed a need for regular training and updates as insufficient NPS-related information is currently received. Conclusions: An improvement within the self-reported knowledge of HCPs on NPS is evident in comparison to previous studies. Continued education of HCPs on NPS is fundamental for the provision of improved harm reduction services, which can enhance overall care for NPS service users.