Acute ischemic stroke (AIS) is one of the leading causes of morbidity worldwide, thus, early recognition is essential to accelerate treatment. The only definite way to diagnose AIS is radiological imaging, which is limited to hospitals. However, two serum neuromarkers, glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1), have been proven as indicators of brain trauma and AIS. We aimed to investigate the potential utility of these markers in distinguishing between large vessel occlusion (LVO) and small vessel occlusion (SVO), considering differences in treatment. Sixty-nine AIS patients were included in our study and divided into LVO and SVO groups based on radiological imaging. Control group consisted of 22 participants without history of neurological disorders. Results showed differences in serum levels of both GFAP and UHC-L1 between all groups; control vs. SVO vs. LVO (GFAP: 30.19 pg/mL vs. 58.6 pg/mL vs. 321.3 pg/mL; UCH-L1: 117.7 pg/mL vs. 251.8 pg/mL vs. 573.1 pg/mL; p < 0.0001), with LVO having the highest values. Other prognostic factors of stroke severity were analyzed and did not correlate with serum biomarkers. In conclusion, a combination of GFAP and UCH-L1 could potentially be a valuable diagnostic tool for differentiating LVO and SVO in AIS patients.