Interactions of 100-nm liposomes prepared from egg yolk phosphatidylcholine and baker’s yeast phosphatidylinositol carrying diglyceride ester conjugates of melphalan (Mlph-liposomes) and methotrexate (MTX-liposomes) in the bilayer with blood plasma proteins were studied with Western blotting. Earlier hemocompatibility tests have demonstrated that the liposomes did not affect main blood cells, but MTX-liposomes, and not Mlph-liposomes, induced complement (C) activation in vitro. Here, we show that introduction of polyethylene glycol conjugate (instead of phosphatidylinositol as a stabilizing lipid) or a targeting carbohydrate conjugate has little effect on interaction of Mlph-liposomes with major C components and apolipoproteins, as well as the total protein binding ability of the liposomes. Liposomes loaded with Mlph prodrug did not trigger fragmentation of C3 protein, the central component of the complement, while MTX-liposomes did so, which agrees with our previous findings. Analysis of MTX-liposome binding with C3 protein and its fragments, regulatory C proteins, and immunoglobulins allowed for the conclusion that MTX-liposomes activate complement via the alternative activation pathway. As shown earlier, the decrease in the prodrug concentration in the bilayer to the level corresponding to MTX low-dose treatment regimen allows avoiding C activation.