Systemic inflammation has been shown to predict poorer chemotherapy response and a decrease in overall survival in cancer patients. While the success of immunotherapy has been well documented in specific cancer such as melanoma, there is a lack of understanding of the complexity of the tumour-immune interactions in other cancers, such as colorectal cancer. A new single-cell technique, mass cytometry, has allowed for the measurements of multiple intracellular markers in a single panel. Our aim was to use this innovative technique to investigate the expression of inflammatory signalling pathways in the major immune subtypes in cancer patients undergoing chemotherapy and identify whether the activation of specific pathways is correlated with their clinical outcomes. A mass cytometry panel was designed to measure 25 surface markers and 10 intracellular inflammatory signalling pathway proteins (including STAT, NF-κB, ERK) using Helios™, a Cytometry by Time-of-Flight (CyTOF) system (Fluidigm). We quantified the expression of the signalling pathway proteins in 7 major immune cells, including B cells, T cells, and myeloid cells, in 10 stage III and IV colorectal cancer patients and 9 age and gender matched healthy volunteers. At baseline, our results show that pP38 and pSTAT3 are significantly decreased compared to the healthy volunteers across the majority of the major immune cell types whereas pSTAT5 is significantly increased, particularly in B cells. Our results also show that phosphorylated markers, such as pERK and pMAPKAPK2, can also be activated following a cycle of chemotherapy and can remain activated throughout the course of patients’ therapy. Relationships between immune profiles and clinical outcomes are currently being explored. The use of mass cytometry has allowed the rapid quantification of the immune signalling pathways in specific immune cell types. This could potentially enable us to predict patient response as well as identify new drug targets to improve patient outcomes.