Multiple sclerosis (MS) is a complex, neurodegenerative and autoimmune disease with a multifactorial etiology. Epigenetic changes can influence the onset and development of the disease, and miRNAs, small RNAs that play a key role in post-transcriptional gene regulation, act as important modulators of the inflammatory process and the central nervous system´s response. The objective of this work is to compare and identify the expression of the miR-143/145 cluster, namely miR-143-5p and miR-145-5p. For this purpose, the expression of two mature miRNAs was determined in the serum of 80 patients with relapsing-remitting multiple sclerosis (RRMS) under different treatments and 60 healthy control subjects (HCS). Total miRNA was isolated from serum and subjected to quantitative PCR analysis (qPCR). In addition, an in silico analysis focused on the molecules affected by the disease was performed. It was found that miR-143-5p is upregulated in patients with RRMS (mean (m)= 0.74) compared to HCS (m = 1.50), while miR-145-5p is significantly downregulated in RRMS (m = 0.32) compared to HCS (m = 2.26). Furthermore, miR-143-5p expression may vary with treatment and exhibit sexual dimorphism, whereas miR-145 expression is primarily pathology-dependent. For in silico analysis, we proposed a new modified C-score that integrates several computational tools. This analysis indicates that GSTM3 and MMP9, which have been previously studied in MS, are the primary targets of miR-143-5p and miR-145-5p, respectively. In conclusion, the differential expression of these miRNAs underscores their possible sensitizing role in pathogenesis, suggesting that these two miRNAs, along with others, may be further explored in unraveling new pathophysiological pathways for treatment response in MS.