Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes.