Objective: This study aimed to determine the standard of gene expression of iNOS in gingival tissue from pregnancy women with and without periodontal disease. Method: Data were expressed as Cycle threshold (Ct) values and relative quantification performed. The comparison between the two groups was performed using the program Rest© 2008. Gingival tissue was collected from an area with presence of periodontal pockets deeper than 4 mm in women with periodontal disease. In women without periodontal disease gingival tissue was collected from adjacent tissue to teeth with indication for extraction. All samples were collected during dental care in a School of Dentistry in Juiz de Fora, in the state of Minas Gerais, Southeast region of Brazil. Samples of gingival tissue (about 54 mg) were immediately immersed in 1.5 mL RNALatter and stored at -20 °C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen), following the manufacturer's recommendations. The first strand was synthesized and cDNA analysis of iNOS was performed by means of Real Time PCR. Results: This study analyzed the expression of iNOS gene into two pools consisting of samples of gingival tissue from these groups. The best concentrations of primer and the target gene cDNA for iNOS and the endogenous controls β-actin and GAPDH were 100 nM–400 ng, 50 nM–100 ng and 200 nM–100 ng, respectively. The amplification efficiency of target gene and endogenous controls was 0.9 and the temperatures of dissociation were 79°C, 86.2ºC and 86.5°C, respectively. There were no peaks related to the amplification primer dimmer or nonspecific products to any genes when analyzing the dissociation curve. Conclusion: The pre-established conditions for the analysis will be able to draw a profile of differential expression for each group.