There has been a rapid grown in the field of tumour immunobiology and in cancer immunotherapies and it is becoming clear that immune cells play many sometimes conflicting roles in the tumour microenvironment. However, obtaining phenotypic information about the various immune cells around the tumour has been a challenge. We present here a methodology for delivering quantitative per-cell marker expression and phenotyping, analogous to that obtained from flow cytometry, but from cells imaged in situ in FFPE tissue sections. This methodology combines: the sequential multi-marker labelling of up to eight antigens using antibodies all of the same species in a single section; automated multispectral imaging (MSI) to remove the problematic FFPE tissue autofluorescence and correct cross-talk between fluorescent channels; and an automated analysis that can quantitate the per-cell marker expression, determine the cellular phenotype, count these cells separately in tissue compartments and provide high-resolution images of their distributions. We present here three examples: the simultaneous labelling, analysis and validation of CD3, CD8 and FOXP3 multiplexed staining in follicular lymphoma; a CD4, CD8, CD20, pan-CK TH, TC and B cell panel in breast cancer; and PD-L1, CD8, CD34 and FOXP3 phenotyping and quantitation in melanoma. Each example will show the application of the multiplexed staining, per-cell quantitation and cellular phenotyping from multispectral images of FFPE tissue sections.