During the past decade, biodegradable polymers or oligopeptides recognized by cell-surface receptors have been shown to increase drug specificity, lowering systemic drug toxicity in contrast to small-size fast-acting drugs. The goal of the present study was to develop anticancer bioconjugates based on chemotactic drug targeting (CDT). These constructs are composed of methotrexate (Mtx) attached to a tuftsin-like peptide carrier through an enzyme-labile pentapeptide spacer (GFLGC) and several copies of a chemotactic targeting moiety (H-TKPR, For-TKPR, H-TKPKG, and Ac-TKPKG). Carriers with targeting moieties in the branches were prepared by solid-phase synthesis using mixed Boc and Fmoc strategies. The drug molecule connected to an enzyme-labile spacer was attached to the branched oligopeptide in solution. In vitro chemotaxis, cellular uptake, and cytotoxicity assays were carried out on the MonoMac6 cell line. The most effective conjugates with H-TKPR or Ac-TKPKG targeting moieties in the branches, which have the most advantageous chemotactic properties, can be internalized rapidly, and these conjugates trigger higher toxic effect than the free drug (Mtx). The results suggest that our tuftsin-based drug delivery systems might be potential candidates for targeting cancer chemotherapy.