Background: There is a need to identify biomarkers which could indicate the occurrence of brain injury in drug abuse.Objectives: We aimed to investigate ubiquitin-C-terminal hydrolase-1 (UCH-L1), a neuronal cell body injury marker, the glial protein S-100 beta (S100β), and the glial fibrillary acidic protein (GFAP) as putative markers for neuronal injury due to cannabis, tramadol, or their combined use. Materials and methods: Rats were treated with cannabis and/or tramadol subcutaneously daily for 6 weeks and UCH-L1, S100β, and GFAP were immunoassayed in the brain and serum.Results: The results are as follows: (i) either cannabis or tramadol increased UCH-L1 and GFAP in the brain, (ii) serum UCH-L1 and GFAP increased by the highest dose of cannabis or tramadol, (iii) there was no additive effect for cannabis and tramadol on UCH-L1 or GFAP level in the brain or serum, (iv) S100β decreased in the brain by 5-20 mg/kg of cannabis and in the serum following 20 mg/kg of cannabis, and (v) S100β levels increased in the brain after 20 mg/kg of tramadol but decreased the brain and serum after both cannabis and tramadol. Cytoplasmic vacuolations, apoptotic cells, and gliosis were observed in the brain tissue of cannabis and/or tramadol-treated rats. Conclusions: These results suggest that changes in UCH-L1, GFAP, or S100β are likely to reflect neurotoxicity and serum levels could be used to detect neuronal damage in chronic users.