Endocytosis is an essential process of eukaryotic cells that facilitates numerous cellular and organismal functions. The formation of vesicles from the plasma membrane serves the internalization of ligands and receptors and leads to their degradation or recycling. A number of distinct mechanisms have been described over the years, several of which are only partially characterized in terms of mechanism and function. These are often referred to as novel endocytic pathways. The pathways differ in their mode of uptake and in their intracellular destination. Here, an overview of the set of cellular proteins that facilitate the different pathways is provided. Further, the approaches to distinguish between the pathways by different modes of perturbation are critically discussed, emphasizing the use of genetic tools such as dominant negative mutant proteins.