The neurotrophin brain-derived neurotrophic factor (BDNF) is extensively expressed in neural cells and other cell types. BDNF displays a support function in both central CNS and peripheral PNS where it is involved in most neuronal mechanisms, from development to neurodegeneration, cellular growth, neuroprotection, survival, and network signaling. In the last decades, decreased levels of BDNF were directly linked to Parkinson’s, Alzheimer’s, and Huntington’s disease, while BDNF upregulation was found in patients affected by epilepsy, autism, and multiple sclerosis. Accordingly, determination of BDNF expression can contribute to gain insight in several pathological models both in vivo and in vitro. In this manuscript, we provide detailed description of the main techniques for BDNF detection in cultured neurons and in brain slices, using different methods such as ELISA, Western blot, and immunofluorescence.