Total knee arthroplasty is a well-established treatment for degenerative joint disease, on the other hand metal ion release of cobalt or chromium and particle formation can trigger intolerance reactions. Biotribological examinations can help to assess the metal ion release in different settings. The purpose of this study was the evaluation of inter-laboratory differences in the metal ion concentration analysis. Samples were generated in a 3+1 station knee wear simulator (EndoLab GmbH, Thansau, Germany) with a medium size Columbus Knee System with or without AS multilayer coating. The wear simulation was performed under highly demanding activity (HDA) profile and samples were taken after 0.5, 5.0, 5.5. and 8.0 million cycles. The samples were blinded and sent to three different laboratories and the content of chromium, cobalt, molybdenum, nickel, and zirconium was assessed by inductively coupled plasma mass spectrometry (ICP-MS). The AS multilayer coating clearly reduced the release of chromium, cobalt and molybdenum. Mean levels were: Chromium 9329.78µg/l ± 985.44 vs 503.75µg/l ± 54.19, cobalt 10419.00µg/l ± 15.517.53 vs 2.60µg/l ± 1.35, molybdenum 2496.33µg/l ± 102.62 vs 2.46µg/l ± 2.31. Interestingly we found especially for nickel and zirconium big inter-laboratory differences in the metal assessment. There were up to 10-fold higher values in comparison of one laboratory to another. The data demonstrate that results of metal ion assessment should be evaluated by interlaboratory comparison and should be critically interpreted.