Patient-derived organoids (PDOs) can model personalised therapy responses. However, current PDO screening technologies cannot reveal drug response mechanisms or study how cells of the tumour microenvironment alter therapeutic performance. To address this, I optimised the TOBis MC platform established in our lab to develop a highly-multiplexed mass cytometry screening platform to measure post-translational modification (PTM) signalling in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at a single-cell resolution. To compare patient- and microenvironment-specific drug responses across thousands of single-cell datasets, a highly-scalable analysis method was developed – Trellis. Trellis enables fast and accurate hierarchical tree-based treatment effect analysis across thousands of distributions. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is patient-specific. Drug-induced apoptosis does not correlate with genotype or clinical staging but does align with cell-intrinsic PTM signalling in PDOs. CAFs protect chemosensitive PDOs by shifting cancer cells into a slow-cycling cell-state and CAF chemoprotection can be reversed by inhibiting YAP.