Transgenic cotton plants are responsible of a greater breakthrough in the field of agricultural biotechnology which focuses on improving plant characteristics such as pest resistance, drought tolerance, and fiber quality. The precise molecular characterization of these genetically transformed plants is fundamental in assessing the insertion, inheritance, and expression of transgenes. The β-glucuronidase (GUS) assay is among the conventional tools to test transgenic cotton that offer a consistent and productive process for determining gene expression. This chapter offers the exploration of the GUS assay using transgenic cotton plants, which may be useful for gene development. The enzyme that gives the blue product is β-glucuronidase, which in turn cleaves its substrate (5-bromo-4-chloro-3-indolyl β-D-glucuronide [X-Gluc]). It signals a change in color which can be seen directly as an indicator for the transgene expression. The histochemical assay significance is based on tissue fixation time, incubation periods, staining, and decolorizing of chlorophyll procedures for reproducible results of putative transgenic lines. It also covers approaches for optimizing assay conditions, such as changing the levels of substrates and incubation times to improve staining clarity and intensity. This chapter provides the step-by-step protocol for initial analysis of transgenic cotton by reporter assay by GUS histochemical staining.