We are currently conducting clinical research on cell sheets for cartilage regeneration. One issue with the future use of chondrocyte sheets as cellular and tissue-based products is quality assessment. Currently, chondrocyte sheets are evaluated using invasive methods that cannot be performed on every sheet produced. We report here on our liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique that allows the noninvasive assessment of every sheet using only 50 I of culture medium. We found that LC-MS/MS could be used to confirm cell sheet viability through the measurement of glucose and glutamine uptake, to estimate extracellular matrix production by measuring serine consumption, to estimate cell kinetics by measuring cytidine and uracil concentrations, and to estimate melanoma inhibitory activity level by measuring pyridoxal concentration. LC-MS/MS may be useful for the noninvasive assessment of products to be used in regenerative medicine. (C) 2018 The Authors. Published by Elsevier Inc.