Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation on ADC stability due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogenous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics and, efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g. PNU-159682, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C and HC-A140C) on THIOMABTM antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.