The expression gene pattern reflects, in part, mechanisms involved in adaptation to environmental conditions. Thus, we established and validated a method that enables relative transcript quantification in different conditions in the lactic acid bacteria Oenococcus oeni, notably in a technological medium. First, we determined an internal control in our conditions by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the SYBR Green I technology. Among the seven presumed housekeeping tested genes, the ldhD gene was retained for further experiments. Then, the PCR reproducibility was verified in our conditions and the comparative critical threshold (2deltadeltaC(T)) method was applied to quantify the transcript level of genes. The quantification of transcript levels of several stress genes already studied in our laboratory by Northern blot after a heat shock and at the entry of stationary phase allowed us to validate this method. RT-qPCR appeared as a powerful tool to study O. oeni response in stress conditions and wine mimetic conditions.