Successful breeding programs optimize time and resources to produce elite lines. Selecting individual plants in the F2 generation is an efficient strategy if the trait is highly heritable and nondestructive methods exist to analyze the seed. Cotton (Gossypium hirsutum L.) seed has limited uses because of gossypol, a toxic compound found in the seed. Gossypol exists in two enantiomeric forms with the (+) less toxic than the (−) form. Reducing gossypol or increasing the (+) enantiomer in the seed would increase the amount that could be fed to livestock, chicken, or fish. Rapid, cost-effective methods were developed to measure (+) and (−) gossypol in the cotyledon (chalazal) half of a seed by high performance liquid chromatography (HPLC) on a reduced scale. Techniques were also developed to propagate the embryo (micropylar) half of the seed. The techniques were used to develop elite lines with varied gossypol levels produced one year earlier than possible with more conventional breeding strategies. The half-seed methods combined with a modified HPLC gossypol assay provided a simple, cost-effective method to breed for modified gossypol content and enantiomer composition. These methods can be combined with other testing or evaluation techniques to further optimize selection efficiency.