Labeling of platelets (PLTs) is required to measure the recovery and survival of transfused PLTs in vivo. Currently a radioactive method is used to label PLTs. However, application of those radiolabeling methods is limited by both safety issues and the inability to isolate transfused PLTs from the circulation. Biotin-labeled PLTs are an attractive nonradioactive option. However, no validated protocol to biotinylate PLTs is currently available for human studies. Six PLT concentrates (PCs) were subaliquoted and biotinylated on Days 1 and 7 of storage. To distinguish the effect of the processing steps from the effects of biotin incubation, two control groups were used: 1) "sham" samples were processed without the biotinylation reagent and 2) control samples were assessed without any processing other than the PC isolation. For the biotinylation procedure, 50 mL of PCs was washed twice and incubated with 5 mg/L biotin for 30 minutes in a closed system. As measures of PLT activation, phosphatidylserine exposure and CD62p expression were assessed. After biotinylation, 98.4% ± 0.9% of PLTs were labeled. PLT counts, pH, and "swirling" were within the range accepted by the Dutch blood bank for standard PLT products. Biotinylated PLTs were more activated compared than controles but not more than sham samples, but were more activated than the controls. We developed a standardized and reproducible protocol according to Good Practice Guidelines standards, for biotin labeling of PLTs for clinical purposes. This method can be applied as nonradioactive alternative assess survival and recovery of transfused PLTs in vivo. © 2019 The Authors. Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.