Proteases play a key role in protein digestion in ticks and other haematophagous insects. Our understanding of blood meal digestion in digestive system of ticks can be very useful for better understanding of basic rules for control of ticks. Cells of the midgut endocytose blood components. Blood proteins uptake by midgut cells, suggesting the presence of proteases in the midgut cells. In this study, proteases which may be present in midgut cells of engorged female ticks (Hyalomma anatolicum anatolicum) have been studied. The midgut tissue from tick was dissociated to cells. The cells placed in a culture plate with special medium. Then cell extract was obtained from the in vitro cultured midgut cells. Then cells were rinsed in hypotonic solution. The cellular suspension was centrifuged and supernatant removed from cell membrane plette. Cell membrane proteins were isolated by Solution 2% of Triton X-100. Proteases assay performed by specific substrates for cytoplasmic proteins and Cell membrane bound proteins. Then, enzyme like trypsin, enzyme like chymotrypsin, carboxypeptidase B, cathepsin C, cathepsin B and cathepsin D were detected. Our results show that enzyme like trypsin is a membrane bound protein, but carboxypeptidase B, cathepsin C, cathepsin B and cathepsin D are included in the cytoplasm of mid-gut cells. There is no enzyme like chymotrypsin in mid-gut cells of tick.