During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell‐based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: (i) demonstrate a correlation between S100B + A1‐positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; (ii) develop an S100B + A1 cell‐based ELISA, and (iii) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high‐osmolarity/BMP4 treatment in monolayer) using qRT‐PCR, immunocytochemistry, or immunohistochemistry. A cell‐based ELISA (CELISA) was developed as a 96‐well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow‐derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1‐positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1‐CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high‐throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets—induced with high‐osmolarity and BMP4 treatment in monolayer—suggests that cell instruction prior to implantation may improve cartilage repair. J. Cell. Physiol. 232: 1559–1570, 2017. © 2016 Wiley Periodicals, Inc. The novel S100B + A1‐CELISA offers a high‐throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and identifying redifferentiation factors/conditions to improve cartilage repair. Using a combination of high‐osmolarity medium and BMP4 treatment uncovered with this assay, we demonstrate that the induction of chondrocyte redifferentiation in monolayer improves their chondrogenic potential in pellets.