The skin is essential for body protection and regulation. Raman spectroscopy (RS) is an effective method for identifying the complex biomolecular composition of the skin. The objectives were to characterize the biomolecular composition of the skin of Wistar rats and to verify homogeneity among groups using RS. 30 seven-week-old male Wistar rats were used. A fragment of 3 cm in diameter of the animal’s skin was removed by excisional surgery. The collected fragment was submitted to RS at five different points with excitation at 830 nm and accumulation for 30 s. The data was pre-processed and PCA was applied. The results showed spectral characteristic of lipoproteins. PCA identified differences in the biochemical composition of the skin among the groups. Six PCs were sufficient to show that the biochemical composition of the tissue was relatively constant among the samples, with differences in some PCs for some groups. PC2 showed statistical difference between G5, having lipids peaks (1260, 1300, 1439 and 1661 cm−1), compared to the other groups and having proteins peaks (1003, 1244 and 1341 cm−1). PC3 and PC4 maintained the lipoproteins characteristics of the biomolecular structure of animal’s skin. Raman peaks of carotenoids (1140, 1143, 1340, 1450, 1440 and 1530 cm−1) were found in PC5 and PC6. RS was effective and characterized the biomolecular composition of rat’s skin. Even with control samples, RS showed differences between groups in the biochemical composition of the skin with the use of PCA.