Cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers capable of metastasising. Proteomic analysis of cSCCs can provide insight into biological processes responsible for metastasis as well as future therapeutic targets and prognostic biomarkers.This study aimed to identify proteins associated with development of metastasis in cSCC.A proteomic-based approach was employed on 105 completely-excised, primary cSCCs, comprising 52 which metastasised (P-M) and 53 which had not metastasised at 5 years post-surgery (P-NM). Formalin-fixed, paraffin-embedded cSCCs were microdissected and subjected to proteomic profiling after one dimensional (1D), and separately two dimensional (2D), liquid chromatography fractionation.A discovery set of 24 P-Ms and 24 P-NMs identified 144 significantly differentially expressed proteins, including 33 proteins identified via both 1D and 2D separation, between P-Ms and P-NMs. Several differentially expressed proteins were also associated with survival in SCCs of other organs. Findings were verified by multiple reaction monitoring on 6 peptides from 2 proteins, Annexin A5 (ANXA5) and Dolichyl-diphosphooligosaccahride-protein glycosyltransferase non-catalytic subunit (DDOST), in the discovery group and validated on a separate cohort (n=57). Increased expression of ANXA5 and DDOST was associated with reduced time to metastasis in cSCC and decreased survival in cervical and oropharyngeal cancer. A prediction model using ANXA5 and DDOST had an area under the curve (AUC) of 0.929 (CI=0.8277-1), an accuracy of 91.18% and higher sensitivity and specificity than cSCC staging systems currently in clinical use.This study highlights that increased expression of two proteins, ANXA5 and DDOST, is significantly associated with poorer clinical outcomes in cSCC.