In this paper, we offer detailed information concerning manipulations with the novel serum-free cell line McCoy-Plovdiv. Guidelines for procedures as trypsinization of the monolayer, subculturing, as well as freezing and thawing conditions are proposed. Our results give us grounds to assume that this is a cell line entirely serum-independent at any step of the process of culturing and preservation. The serum-free cell line McCoy-Plovdiv enriches the collection of mammalian serum-free cell lines. The easier cultivation, the lower expenses and the higher sensitivity in comparison with the serum-supplemented McCoy cells are discussed as possibilities for broad applications of McCoy-Plovdiv cells in different types of laboratory investigations.