Assessing in situ microbial abilities of soils to degrade pesticides is of great interest giving insight in soil filtering capability, which is a key ecosystem function limiting pollution of groundwater. Quantification of pesticide-degrading gene expression by reverse transcription quantitative PCR (RT-qPCR) was tested as a suitable indicator to monitor pesticide biodegradation performances in soil. RNA extraction protocol was optimized to enhance the yield and quality of RNA recovered from soil samples to perform RT-qPCR assays. As a model, the activity of atrazine-degrading communities was monitored using RT-qPCRs to estimate the level of expression of atzD in five agricultural soils showing different atrazine mineralization abilities. Interestingly, the relative abundance of atzD mRNA copy numbers was positively correlated to the maximum rate and to the maximal amount of atrazine mineralized. Our findings indicate that the quantification of pesticide-degrading gene expression may be suitable to assess biodegradation performance in soil and monitor natural attenuation of pesticide.