The search for new chemical entities as potential TLRs modulators is an ongoing process, especially because relatively few compounds with therapeutic potential have been tested in clinical trials. Additionally, the use of a strategy involving the TLRs as a driving force for the design of next-generation vaccines has become increasingly popular recently. Since different types of modulators (small-molecule or part of the vaccines, e.g. epitopes), requires various methods and techniques for their identification, we reviewed both classes separately.