In this chapter we focus for characterizing the RNA interference (RNAi) tobacco plants and detecting existence of the small interfering RNAs (siRNAs). RNAi, the most powerful gene silencing approach that engages in introducing double-stranded RNA (dsRNA) homologous to the target gene. The popular model plant system, tobacco, is employed for studying RNAi because of easy transformation as well as fast growth. This chapter emphasizes the steps that include total RNA extraction, cDNA synthesis, qRT-PCR amplification, as well as northern blotting. To segregate the DNA contamination from isolated total RNA extracted using TRIzol method, DNase treatment was carried out. PCR amplification was performed to check for the target gene and housekeeping gene. Northern blot experiment was done to examine the existence of siRNAs. The experiment worked in that RNAi-mediated gene silencing occurred in the tobacco plants, as proven by lower expression of the target gene and detection of siRNAs. This research offers a great protocol for the analysis of RNAi tobacco plants and the detection of siRNAs and can be utilized in many different research fields, such as plant biotechnology and functional genomics.