Background. Alternative splicing (AS) expands transcriptomic and proteomic diversity, generating protein isoforms with specific or opposing functions. Aberrant AS contributes to glioblastoma (GBM) development and aggressiveness. Consequently, isoform-specific therapeutic approaches are emerging as promising strategies for targeted cancer treatment. Building upon TCGA datasets and previous functional studies of ITSN1 (ITSN1-L, ITSN1-S) and CHI3L1 (CHI3L1-FL, CHI3L1-del8) isoforms in glioma cell lines, the present study aimed to investigate their distribution in human GBM specimens. Methods. Patient samples of IDH-wt GBM were collected following informed consent and in accordance with ethical regulations. Tumor tissues (n = 35) and matched adjacent non-tumorous brain tissues (n = 10) were obtained from patients undergoing surgical resection.Transcript levels were quantified by RT-qPCR using isoform-specific primers designed in our laboratory. The RPLP0 and GAPDH were used for normalization. Diagnostic performance was evaluated by ROC curve analysis. The ITSN1-L/ITSN1-S ratio was calculated from their normalized expression levels. Results. Statistically significant alterations in isoform expression were observed in GBM samples. mRNA levels of both CHI3L1 isoforms were increased in tumors vs control tissues (AUC-del8 = 0.9298, 95% CI 0,8495 to 1,000; AUC-FL = 0.9444, 95% CI 0,8371 to 1,000). In contrast, both ITSN1 transcripts were significantly decreased in GBM (AUC-S = 0.9402, 95% CI 0,9351 to 1,000; AUC-L = 0.9658, 95% CI 0,9612 to 1,000). Moreover, reduced relative abundance of the ITSN1- L vs ITSN1-S was detected in tumor tissues (AUC = 0.9231, 95% CI 0,8779 to 1,000). Conclusions. This pilot study demonstrates altered regulation of the major ITSN1 and CHI3L1 transcript isoforms in GBM. The pronounced reduction of ITSN1-L vs ITSN1-S supports previous reports suggesting a stronger tumor-suppressive role for the ITSN1-L. These results highlight the importance of AS in GBM biology and suggest the potential of specific isoforms as diagnostic biomarkers and therapeutic targets. Funding. This work was supported by a grant from the NAS of Ukraine for research laboratories of young scientists conducting studies in priority areas of science and technology development (2025—2026, #0125U001298) and IMBG Simons Foundation Grant for Ukrainian institutions (№ SFI-PD-Ukraine-00017453).