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Research on the knowledge of PrEP in healthcare workers including third sector workers is limited, and their knowledge will be vital to future national rollout. The aim of this study was to design and evaluate a teaching toolkit on PrEP to educate healthcare professionals and third-sector workers.A 20 minute powerpoint teaching toolkit was designed and delivered to sexual health workers, third sector workers and medical students. A questionnaire was used to evaluate the toolkit, including perceived knowledge pre-toolkit, immediate post-toolkit, and >1-week post-toolkit.42 participants took part in teaching sessions. There was a 36% increase in mean perceived participant knowledge scores (maximum = 25) immediately after teaching (23.69), and a 26% increase >1-week after teaching (21.93) – when both are compared with a prior mean score of 17.45. This change in perceived knowledge increased significantly both immediately post and >1-week post when compared with pre-toolkit (Z = -5.351, p = <0.001; Z = -3.189, p = 0.001). Immediately after, 42/42 (100%) participants agreed they had some knowledge of the monitoring and tests for PrEP in comparison to 21/42 (50%) pre-teaching (Z = -4.753, p = <0.001). Overall 39/42 (93%) of participants strongly agreed it provided a good overview of PrEP, with 35/42 (84%) thinking it would help them to provide answers to those seeking to use PrEP.Perceived knowledge of PrEP increased following toolkit use and importantly was sustained >1-week post-toolkit when compared with prior knowledge. Toolkits such as this can help educate future PrEP advocates.