The degree of stability of antibody–drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody–drug conjugates (ADC) in vivo. Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC–based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC–based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958–70. ©2016 AACR.