Antibody-drug conjugates (ADCs) are cancer therapeutics that harness the targeting abilities of monoclonal antibodies to deliver small-molecule drugs directly to tumors. Characterization of such conjugates involves determining the drug-to-antibody ratio—how many drug molecules are attached to each antibody. The standard method for determining the drug-to-antibody ratio is hydrophobic interaction chromatography, but that separation method isn’t compatible with mass-spectrometric detection. Now, a team led by Mary J. Wirth of Purdue University, working with researchers at AbbVie, reports a method called native reversed-phase liquid chromatography, which allows the characterization of intact ADCs by mass spectrometry (Anal. Chem. 2019, DOI: 10.1021/acs.analchem.8b04699). The keys to the method are using a low concentration of an MS-compatible salt, a mild organic additive, and a low-hydrophobicity stationary phase, so that ADCs can traverse the column without getting stuck or falling apart. By combining the new separation method with mass spectrometry, the researchers detected ADCs with various numbers of attached drug molecules. They demonstrated the method using a model ADC from AbbVie and a commercial ADC, brentuximab vedotin, from Seattle Genetics. A drawback of the method is that it doesn’t work well for the antibody with no drug attached.