Quantitative gene expression analysis is widely used to evaluate the expression of specific tissue markers. To obtain reliable data it is essential to select stable housekeeping genes whose expression is not influenced by the anatomical origin of cells or by the culture conditions. No studies have evaluated housekeeping gene stability in intervertebral disc (IVD) cells and only few studies using cartilaginous endplate (CEP) and articular cartilage (AC) cells are present in the literature. We analysed the stability of four candidate housekeeping genes (GAPDH,TBP,YWHAZandRPL13A) in human cells isolated from nucleus pulposus (NP) and annulus fibrosus (AF), CEP and AC. Cell isolation, expansion, cryoconservation, and differentiation in 3D pellets were tested. GeNorm, NormFinder, BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability. In each cell population,TBPalone or combined withYWHAZwas identified as the best normaliser in both monolayer and 3D pellets.GAPDHwas the best performer only for AC cells in monolayer. In most culture conditions considering groups of two or more cell types,TBPwas the most stable andYWHAZwas the second choice.GAPDHwas the best performer only in 3D pellets with factors for AC and AF combined with CEP cells.RPL13Awas the most stable only for AF with CEP cells at isolation. Our findings will be useful to properly design the experimental set-up of studies involving IVD, CEP or AC cells in different culture conditions, in order to obtain accurate and high quality data from quantitative gene expression analysis.