Recently, it has been suggested that cellular senescence is associated with stem cell exhaustion, which reduces the regenerative potential of tissues and contributes to aging and age-related diseases. Mesenchymal stem cells (MSCs) attract a large amount of attention in stem cell research and regeneration medicine because they possess multiple advantages and senescent MSCs could be one of the most useful stem cell models in aging studies. It is important to quantitatively evaluate senescence markers to both identify and study the mechanisms involved in MSC senescence. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the most widely used tool to quantify the mRNA levels of markers. However, no report has demonstrated the optimal reference genes that should be used to normalize RT-qPCR in senescence studies of MSCs. In this study, we compared 16 commonly used reference genes (GAPDH, ACTB, RPL13A, TBP, B2M, GUSB, RPLPO, YWHAZ, RPS18, EEF1A1, ATP5F1, HPRT1, PGK1, TFRC, UBC, and PPIA) in proliferating or replicative-senescent human adipose-derived MSCs (hAD-MSCs) that were isolated from seven healthy donors aged 29-59 years old. Three algorithms (geNorm, NormFinder, and BestKeeper) were used to determine the most optimal reference gene. The results showed that PPIA exhibited the most stable expression during senescence, while the widely used ACTB exhibited the lowest stability. We also confirmed that different reference genes lead to different evaluations of senescence markers. Our work ensures that results obtained from senescence studies of hAD-MSCs will be appropriately evaluated in both basic research and clinical trials.