Increasing antibiotic resistance of clinically relevant microorganisms poses important risks to human health. Recently published research has suggested that the deposition of incompletely metabolized antibiotics in fecal wastes originating from human (via wastewater, leaky septics) and animal (via feedlot runoff, biosolid application) sources is contributing to antibiotic resistance in environmental microorganisms. Further, it has been proposed that antibiotic micropollutants present a potential pathway for increased bacterial resistance in clinical settings. Many antibiotics used for treating infections are produced by environmental microorganisms, meaning that resistance genes must also have emerged in non-clinical habitats. Thus, the methods for assessing elevations in antibiotic resistance in environmental isolates must also account for levels of pre-existing natural resistance. This talk will discuss the strengths and drawbacks of methods currently used to assess antibiotic resistance in environmental (soil, water, manure) samples, including microdilution methods, real-time PCR for quantification of resistance genes, and antibiotic disc diffusion assays. Results from a study examining temporal shifts in bacterial antibiotic resistance in wastewater bioreactors will be presented to illustrate the potential for different methods to produce contrasting results. Finally, “best practices” methods, that consider sources of error and background resistance levels, will be proposed for research studies assessing the development of resistance in environmental microorganisms.