Breeding and quality assurance programs need accurate and cost effective methods to test for the presence of target and non-target genes in transgenic cotton varieties. The objective of this paper is to detail the Roundup Ready® (RR) Rolled Towel Bioassay procedure for cotton seed and demonstrate the reliability of this test in identifying the RR trait in cotton seed. Planting media is prepared by adding 2250 ml of a 0.30% Roundup Ultra® solution to 1000 g of germination towels. Fifty seeds are planted in germination towels. Towel rolls are placed in a vented plastic container, and then vertically positioned in a 30°C germinator for seven days. Seedlings are evaluated as either Roundup tolerant, susceptible, dead, or hard seed. Roundup tolerant seedlings produce normal seedlings with all essential structures, although growth is usually inhibited by 25-30% compared to a standard germination test. Susceptible seedlings have a shortened hypocotyl-radicle length with characteristic black lesions on hypocotyls. RR Rolled Towel Bioassay results averaged 99.80% purity compared to 99.84% for RR lateral flow strip tests, across 678 truckload samples. The RR Rolled Towel Bioassay is an accurate and cost effective way to detect the presence of the RR trait in cotton seed. Future work will include a referee project to determine repeatability between laboratories and error rates.