Purpose: Dental implant manufacturers recommend healing abutments (HA) be used for single-patient use; however, reuse on multiple patients following decontamination and sterilization is common. This study aims to evaluate four decontamination strategies utilizing enzymatic agents, available in most clinical settings, to determine the level to which biomaterial can be removed in a group of previously used HA (uHA). Secondly, to determine the degree to which the decontaminated HA are capable of inducing an inflammatory response in-vitro compared to new, never used HA. Materials and Methods: Fifty HA were collected following 2-4 weeks of intraoral use and distributed randomly into 5 test groups (Group A-E; n = 10/group). Group A: Enzymatic cleaner foam + Autoclave; Group B: Ultrasonic bath with enzymatic cleaner + Autoclave; Group C: Prophy jet + Enzymatic cleaner foam + Autoclave; Group D: Prophy jet + ultrasonic bath with enzymatic cleaner + Autoclave; Group E: Prophy jet + Autoclave. Ten new, sterile HA served as controls (Group “Control”). Residual protein concentration was determined by a Micro BCA protein assay while HA from each group were stained with Phloxine B and macroscopically examined for the presence of debris. To examine the inflammatory potential, human primary macrophages were exposed to HA and supernatant levels of 9 cytokines/chemokines profiles were analyzed using a multiplex bead assay. Results: All test groups presented with differences in the degree of visual decontamination compared to Controls, with Groups D and E displaying the most effective surface debris removaland reduced protein concentration. Of the detoxification strategies, Groups D and E removed the greatest biomaterial while least effective was Group A. However, compared to Controls, multiplex assays revealed high levels of inflammatory cytokine secretion up to 5 days from all Test Groups (A-E) irrespective of the decontamination method used. Conclusion: Our study found that compared to new, never used HA, decontamination of uHA utilizing enzymatic cleaners failed to reestablish inert HA surfaces and prevent an inflammatory immune response in-vitro. Clinicians should not reuse HA even after attempts to decontaminate and sterilize HA surfaces.