Tumors of the head and neck (HNSCC) are the sixth most diagnosed tumors in the world. Patients diagnosed with HNSCC have a poor prognosis and limited treatment options. Therapies targeting specific targets within the tumor are still in development, which is why it is necessary to conduct research focused on a better understanding of the biology of HNSCC, as well as the identification of new biomarkers for early disease prediction and as new targets for targeted therapies. Many studies confirm that the Hedgehog-GLI (HH-GLI) signaling pathway is one of the main factors necessary for the development and maintenance of HNSCC. The HH-GLI signaling pathway is otherwise involved in embryogenesis, maintenance of stem cell, and tissue regeneration. Abnormal activation of the HH-GLI signaling pathway is associated with the tumorigenesis of numerous tumors. This is enabled via transcription factors GLI1, GLI2, and GLI3 who promote the overexpression of pro-oncogenic genes, many of which remain unidentified. The identification and validation of these genes will enable a better understanding of the biology of HNSCC. For this purpose, I conducted two independent experiments on two HNSCC cell lines. In one experiment, cells were treated with HH-GLI signaling pathway inhibitors, and in the other, they were transfected with plasmid vectors encoding GLI3 constructs. Analysis of the quantitative real-time PCR results confirmed the regulation of 6 genes by the transcription factor GLI3, making them prime targets for new therapeutic approaches.