Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are neurotrophic growth factors, signaling primarily via the tropomyosin receptor kinase B (TrkB). Here, we present a protocol for producing BDNF and NT4 in their pro and active form in Escherichia coli. We describe steps for E. coli expression, oxidative in vitro folding from inclusion bodies, and purification. We then detail procedures for proteolytic activation and characterization of the purified neurotrophins. For complete details on the use and execution of this protocol, please refer to Holzner et al.(1).