Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases. Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment. The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients. A targeted Selected Reaction Monitoring (SRM) assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein. Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank. The calibration curve using peak area ratio (heavy/light) versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences. In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues. Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.