AbstractObjective:The gold standard for hand hygiene (HH) while wearing gloves requires removing gloves, performing HH, and donning new gloves between WHO moments. The novel strategy of applying alcohol-based hand rub (ABHR) directly to gloved hands might be effective and efficient.Design:A mixed-method, multicenter, 3-arm, randomized trial.Setting:Adult and pediatric medical-surgical, intermediate, and intensive care units at 4 hospitals.Participants:Healthcare personnel (HCP).Interventions:HCP were randomized to 3 groups: ABHR applied directly to gloved hands, the current standard, or usual care.Methods:Gloved hands were sampled via direct imprint. Gold-standard and usual-care arms were compared with the ABHR intervention.Results:Bacteria were identified on gloved hands after 432 (67.4%) of 641 observations in the gold-standard arm versus 548 (82.8%) of 662 observations in the intervention arm (P < .01). HH required a mean of 14 seconds in the intervention and a mean of 28.7 seconds in the gold-standard arm (P < .01). Bacteria were identified on gloved hands after 133 (98.5%) of 135 observations in the usual-care arm versus 173 (76.6%) of 226 observations in the intervention arm (P < .01). Of 331 gloves tested 6 (1.8%) were found to have microperforations; all were identified in the intervention arm [6 (2.9%) of 205].Conclusions:Compared with usual care, contamination of gloved hands was significantly reduced by applying ABHR directly to gloved hands but statistically higher than the gold standard. Given time savings and microbiological benefit over usual care and lack of feasibility of adhering to the gold standard, the Centers for Disease Control and Prevention and the World Health Organization should consider advising HCP to decontaminate gloved hands with ABHR when HH moments arise during single-patient encounters.Trial Registration: NCT03445676.