The objective of this study was to assess the impact of incorporating vermiculite into germination tests for both chemically treated and untreated stored cotton seeds. To that end, two independent experiments were conducted using cotton seeds of the cultivar DP1746B2RF under chemical treatment with a mixture of fungicide, insecticides, and nematicide; and both experiments were set up in a completely randomized design with four replications. In Experiment 1, the objective was to determine the ideal water volume for moistening the vermiculite. Four water volumes were used: 1.5, 2.0, 2.5, and 3.0 times the weight of the dry vermiculite. Experiment 2 was conducted in a 2 × 2 × 2 triple factorial arrangement consisting of two substrates (germitest paper and germitest paper + vermiculite), two treatments (chemically treated seeds and untreated seeds), and two storage periods (0 and 150 days). First germination count and germination were evaluated in the two experiments. From the results found in Experiment 1, the water volume corresponding to 3.0 times the weight of the vermiculite provided the best conditions for germination. In Experiment 2, the most appropriate substrate for conducting the germination test of untreated and chemically treated cotton seeds is vermiculite, which led to rapid germination and uniform seedling development.