Antibody-drug conjugates (ADCs) are being designed and used as highly targeted cancer therapies, with five approved by the FDA and over sixty in clinical trials. Building on the success of antibody therapies, ADCs enable highly specific delivery of a toxic payload to a target tumour cell. LifeArc has previous experience in the successful humanisation of antibodies for clinical use, Keytruda, Entyvio, Actemra and Tysabri. In order to build upon this expertise and extend the therapeutic approaches, LifeArc has several ADC programmes in the oncology and non-oncology space. As part of this portfolio, in-house capability has been established and a number of cell-based assays have been developed to identify candidate ADCs. A case study will be presented that outlines the screening process used to characterise the capacity of candidate antibodies to bind, internalise and induce cell death in HEK293 cells overexpressing the receptor of interest. Critical to the success of an ADC programme is the development of effective methodologies to screen for candidate internalisation to the lysosome, where the linker will be cleaved to release the attached cytotoxin. In order to facilitate high-throughput analysis of hybridoma supernatant and humanised variant internalisation LifeArc has developed robust cell-based assays with the IncuCyte S3 Live-Cell Analysis System and pH sensitive dyes which fluoresce in acidic lysosomes and endosomes. Following rapid evaluation with the IncuCyte, deeper insight into the intracellular trafficking of promising candidates was gained with the IN Cell Analyzer 6500 HS high content analysis system. When coupled with intuitive analysis workflows to evaluate antibody co-localisation with the lysosome, the sophisticated sensitivity of this high-content imager has allowed further insight into the profile of promising drug candidates and has facilitated the validation of this receptor as an ADC target.