To date no tumour specific biomarkers for vulva cancer have been identified and the initiation and progression of the disease is poorly characterised and understood. Here we present an examination of vulva cancer progression in a cohort of 4 patients by the analysis of healthy tissue, vulvar intraepithelial neoplasia stage III (VINIII), vulvar squamous cell carcinoma (VSCC) and inguinal lymph node metastastases by MALDI mass spectrometry imaging (MALDI-MSI). 1821 peak groups were detected in the MALDI-MSI experiments, of which 567 were found to have different distributions of signal to noise ratios across the 4 tissue types according to a Kruskal-Wallis test (P≤0.01). Potential peptide identifications for the 567 peak groups were obtained by matching the MALDI-MSI data back to standard nanoLC-ESI-MS/MS data. Six of the highest intensity differentially expressed MALDI-MSI peak groups were targeted for identification by in situ MALDI MS/MS. The 6 in situ sequenced peptides matched back to 5 proteins, with one peptide identified from Histone H2A, Haemoglobin subunit β, Histone H4, and Cytoskeletal 10, and 2 unique peptides identified from β-Actin. In order to validate the MALDI-MSI results the spatial expression pattern and differential expression of β-Actin was further verified by immunohistochemistry and data independent nanoLC-ESI-MS/MS.