Loss of large, vacuolated notochordal cells (NCs) from the human intervertebral disc (IVD) is thought to initiate degeneration and associated back pain. It is therefore hypothesised that implantation of NCs may halt or reverse degeneration and thus relieve back pain. However, NCs are lost in early childhood, therefore iPSCs differentiation to NCs offers a clinically-viable cell source. Here we aimed to characterise the proteomic profile of the human foetal NCs, versus surrounding annulus fibrosus (AF) cells. FACS sorting for CD24, a known NC marker, was used to isolate NC (CD24+) and AF (CD24-) cells from microdissected human IVDs (14-15 weeks post-conception, n=3). Following iTRAQ isobaric tagging, TripleTOF mass spectrometry and subsequent bioinformatics, differential protein expression was validated by immunofluorescence. Our study revealed 100 up-regulated and 8 down-regulated proteins in the CD24+ population (-1.5≥FC≥1.5, P≤0.1), including known (e.g. keratins 8 and 19) and novel phenotypic markers. Ingenuity Pathway Analysis (IPA) revealed pathways known to play an important role in NCs and IVD homeostasis, such as NRF2-mediated oxidative stress response and caveolar-mediated endocytosis, and putative upstream regulators known to be active in NCs e.g. TGFβ1 and SMAD3. The same analysis predicted many previously unknown proteins, pathways and regulators to be active or repressed in NCs. Together, these results validate our method as a powerful tool for isolation and proteomic analysis of foetal NC cells and reveal novel proteins and pathways of potential use in the development of future strategies for the study and treatment of IVD degeneration, including in the development of protocols to direct differentiation of pluripotent stem cells towards NCs.