Microplastic research started at the turn of the millennium and is of growing interest, as microplastics have the potential to affect a whole range of organisms, from the base of the food web to top predators, including humans. To date, most studies are initial assessments of microplastic abundances for a certain area, thereby generally distinguishing three different sampling matrices: water, sediment and biota samples. Those descriptive studies are important to get a first impression of the extent of the problem, but for a proper risk assessment of ecosystems and their inhabitants, analytical studies of microplastic fluxes, sources, sinks, and transportation pathways are of utmost importance. Moreover, to gain insight into the effects microplastics might have on biota, it is crucial to identify realistic environmental concentrations of microplastics. Thus, profound knowledge about the effects of microplastics on biota is still scarce. Effects can vary regarding habitat, functional group of the organism, and polymer type for example, making it difficult to find quick answers to the many open questions. In addition, microplastic research is accompanied by many methodological challenges that need to be overcome first to assess the impact of microplastics on aquatic systems. Thereby, a development of standardized operational protocols (SOPs) is a pre-requisite for comparability among studies. Since SOPs are still lacking and new methods are developed or optimized very frequently, the aim of this chapter is to point out the most crucial challenges in microplastic research and to gather the most recent promising methods used to quantify environmental concentrations of microplastics and effect studies.