Antibody-drug conjugates (ADCs) are an established modality for the tissue-specific delivery of chemotherapeutics. However, due to the hydrophobic nature of many cytotoxic payloads, challenges remain in developing chemically stable ADCs with high drug loading. In previous studies, payload structure, unique stimuli-responsive chemistries, and PEGylated cross-linkers have been used to decrease ADC hydrophobicity. In this work, we investigate the effect of a new parameter, cross-linker sequence. A support-free synthesis of PEGylated, sequence-defined cross-linkers was developed and applied to the synthesis of three constitutionally isomeric ADCs containing PEG side chains and a monomethyl auristatin E payload. Placement of PEG side chains distally from the payload was found to yield an ADC with altered hydrophilicity, antigen binding, and in vitro potency. This work establishes a versatile method for synthesizing multifunctional cross-linkers and identifies cross-linker sequence as a new handle for modulating the performance of ADCs.