We used three label-free minimally invasive methods to characterize individual cells derived from primary and secondary tumours from the same patient, and of the same type – colorectal. Raman spectroscopy distinguished cells by their biochemical ‘fingerprint’ in a vibrational spectrum with 100% accuracy, and revealed that the primary cell line contains more lipids and alpha-helix proteins, whereas the secondary cell line contains more porphyrins and beta-sheet proteins. Stimulated Raman scattering (SRS) microscopy distinguished cells in chemically-specific images of CH₂ bonds which revealed lipid droplets in secondary tumour cells. Atomic force microscopy (AFM) was used to distinguish cells with 80% accuracy by measuring their elasticity – secondary tumour cells (SW620) are around 3 times softer than primary ones (SW480). As well as characterizing the physical and biochemical differences between cell lines in vitro, these techniques offer three novel methods which could potentially be used for diagnosis – to assign a tumour as primary or secondary.