Raman spectroscopy can be a useful tool for performing non-destructive subsurface measurements, allowing for investigations on intact foods that would otherwise require food products to be prepared for analysis, thus creating food waste, and often requiring the addition of chemicals. To overcome this, we have developed a method to measure the concentration of pigment (astaxanthin) and fatty acids in whole, unfilleted Atlantic salmon through the skin using defocused Raman spectroscopy. Subsurface measurements were performed across different regions of Atlantic salmon. PLS regression of the measured spectra gave a moderate correlation between Raman bands and astaxanthin concentration (R2 = 0.5797) and good correlations for fatty acid concentrations (total, saturated, monounsaturated, polyunsaturated, trans; R2 = 0.7457 to 0.8249). This work shows that subsurface Raman measurements can provide useful quantitative information regarding important quality parameters (pigment and fatty acid concentrations) that can aid with production in aquaculture.