Summary Objective The development of a reliable high-throughput transfection protocol for primary human articular chondrocytes. Methods Primary human chondrocytes were isolated from adult knee cartilage by an optimized enzymatic digestion protocol and cultivated in high-density monolayer culture for 3–5 days. Isolated chondrocytes were transfected with a green fluorescent protein (GFP)-expressing reporter construct using amaxa's Nucleofector 96-well Shuttle® System. Transfection efficiencies were measured by fluorescence activated cell sorting and cell viability was determined by an adenosine-5′-triphosphate (ATP) assay. siRNA oligonucleotides (against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were transfected into the cells using the optimized nucleofection protocol and mRNA knockdown values were determined by a branched-DNA assay. Results Transfection efficiencies of more than 70% of surviving cells were achieved routinely with the nucleofection protocol presented in this article. Cell viability 24 h post transfection was around 80%. The cell number used per transfection was reduced to 2 × 105 per sample. In addition, the protocol proved to be well suited for the transfer of siRNA molecules into primary human chondrocytes with suppression rates on the mRNA level of more than 95% (for GAPDH). Conclusions We present the successful use of nucleofection on primary human chondrocytes using a microtiter plate compatible format that for the first time allows the efficient transfection of up to 96 samples in parallel. The optimized nucleofection protocol is offering maximum substrate flexibility by allowing transfer of DNA and siRNA oligonucleotides with the same set of parameters. Moreover, the transfection procedure requires substantially lower cell numbers than single cuvette protocols and is therefore perfectly suited for applications requiring multiple experimental replicates.