Drought stress is one of the most common environmental abiotic stress that affects the overall growth and development of plants. Comparative analysis was carried out between the transgenic plants (Gossypium hirsutum) inherited a gene related to small heat shock protein gene (sHSP) family (GHSP-26) and wild type (non-transgenic plants under different morphological, physiological and biochemical level. The GHSP-26 gene was isolated from the Gossypium arboreum which is one of the most tolerates species of cotton to different stress condition. In this study we also identified and analysed drought responsive genes in both genotypes with the help of Real time PCR. To carry out this task, RNA was extracted from the promising lines under drought stress from which cDNA was synthesized. This cDNA was used in Real Time PCR for the Relative quantification of our genes of interest. Here we also provide the detail protocols of the parameters required for the screening of the drought tolerance genotypes.