Although TRAIL (tumor-necrosis-factor (TNF)-related apoptosis-inducing ligand) has been considered a promising broad-spectrum antitumor agent, its further application was limited by poor drug delivery and TRAIL-resistant tumors. A three-step drug delivery strategy was applied to TRAIL for solving these two obstacles in the form of PEG-TRAIL-MMAE (Monomethyl Auristatin E). PEGylation of TRAIL in the first step was carried out to improve its in vivo pharmacokinetics, while the interaction between TRAIL conjugates with death receptors in the second step was designed to activate the TRAIL extrinsic apoptosis pathway, and the further release of MMAE from the lysosome was the third step for introducing another apoptosis pathway to overcome TRAIL resistance in some tumors. Herein, in order to reach a balance among the three steps, the PEG/MMAE ratio was optimized for PEG-TRAIL-MMAE conjugates. PEG-TRAIL-MMAE conjugates with various PEG/MMAE ratios were prepared and compared with each other regarding their pharmacokinetics (PK) and pharmacodynamics (PD). As a result, PEG-TRAIL-MMAE conjugates with a PEG/MMAE ratio of 1:2 showed prolonged half-life in rats (6.8 h), and the best antitumor activity in vitro (IC50 0.31 nM) and in vivo while no sign of toxicity in xenograft models, suggesting it as a promising multistep drug delivery and antitumor strategy after optimization.