Technical Abstract: The environmental loading of steroid hormones contained in dairy wastes may cause a potential adversely affect on the aquatic species. This work was to investigate the profile of steroid hormones in a typical dairy waste operation system and assess the potential risk of hormone contaminations resulting from land application. Simple and quantitative methods were developed to monitor low-level steroid hormones in complex matrix samples. The preparation method for wastewater analysis consists of solid phase extraction and purification steps, which allows removing the sample matrix interference and achieving low detection limits for the quantification of hormones of interest. Precision of the method was high and the average recoveries were >80% for all steroid hormones except medroxyprogesterone, indicating that the methods are satisfactory for the monitoring of dairy wastes involving solid and liquid samples. The methods developed were applied to the survey of hormones in dairy wastes. The endogenous hormones 17-[alpha]-estradiol, 17-[beta]-estradiol, and estrone were detected in the dairy wastewater and lagoon water. The high concentration of 17'-estradiol in fresh dairy wastewater rapidly decreased along the sewage lane because the chemical can readily transform to its metabolite, estrone. The levels of total steroid hormones in the lagoon water were approximately 1~3 orders of magnitude lower than those in the dairy wastewater. The use of sequencing lagoons may remove the hormones and minimize their loads to the environment from land application. In fresh and piled solid manure samples, four steroid hormones were quantified with relative high concentrations. The piling of solid dairy wastes is a good and easy agriculture practice to reduce the hormone load because steroid contaminants may be readily degraded by manure microorganisms.