The Hedgehog GLI (HH GLI) signaling pathway plays a crucial role in the embryonic development of humans and other mammals. In the adult organism, its activation is limited to the stem cells maintenance and wound healing processes. However, dysregulation of GLI transcription factors in this pathway can lead to aberrant signaling, often contributing to the development of cancers, such as head and neck squamous cell carcinoma (HNSCC). HNSCC encompasses tumors arising in various regions, including the oral cavity, larynx, pharynx (upper, mi ddle, and lower parts), and s alivary glands. It constitutes approximately 6% of all cancers and is associated with a particularly high mortality rate. This research aimed to study tumor cells by creating primary cell lines. We compared the HH GLI pathway genes in these cell lines with those in the tumors they originated from . We also tested certain inhibitors on a HH GLI pathway to see how they affected expression of genes and proteins. We also compared our results with another study on commercial cell line s. The inhibitors, like LiCl, GANT61, and ATO, were found to impact the HH GLI pathway differently. Specifically, LiCl was observed to activate GSK3 β phosphorylation in the cytoplasm, while GANT61 and ATO, when located in the cell nucleus , act directly towards influencing transcription factors . Understanding and manipulating these molecular mechanisms could pave the way for innovative therapeutic approaches in fighting cancers associated with HH GLI dysregulation.