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Leech therapy has been utilized in modern and traditional medicine. Leech saliva contains versatile peptides and molecules that can exert anti-microbial, anti-inflammatory, anti-coagulant, and analgesic activities on the patients. The active components and molecular mechanism of action of these components should be deciphered properly in order to generate biotechnological drug candidates by recombinant production of the leech saliva peptides. In our study, we conducted LC–MS/MS and proteomics analysis on the lyophilized leech saliva extract to determine the components of it. Moreover, this extract was tested on the in vitro-activated macrophages. The extract decreased the production of the pro-inflammatory cytokines by the activated mammalian macrophages compared to the positive control groups. These results suggest that the lyophilized leech saliva can be utilized as an anti-inflammatory biotechnological drug candidate against inflammatory and autoimmune disorders. In vitro studies will be conducted to further decipher the most active ingredients in the leech saliva. These active components will be tested on the animal models of the inflammatory and autoimmune disorders to show their drug potential. [ABSTRACT FROM AUTHOR]