INTRODUCTION: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common malignancy in the world. High mortality and severe complications are critical features of head and neck cancer. Changes in intracellular signaling pathways are a general tumor formation and progression mechanism. Due to the objectivity that the PI3K pathway plays a critical role in HNSCC. the negative regulators involved in this pathway such as GRHL3, PHLDA3, which have been reported to reduce their expression in malignancies, can achieve significant results in the detection, prognosis, and targeted treatment of HNSCC if changes in transcriptome, proteome, and methylation levels of these genes are observed. METHOD: 45 fresh head and neck cancer cells and 45 control samples were collected. Protein expression was also studied using Western blot. Additionally, promoter methylation was investigated using the qMSP method to observe changes in the regulatory regions. RESULTS: The results indicate a significant decrease in GRHL3 expression and a significant increase in PHLDA3 expression. Notably, these expression changes were not confirmed at the protein level. Additionally, methylation analysis revealed hypermethylation of the promoter region in GRHL3 and hypomethylation in PHLDA3. CONCLUSION: This study is the first to examine the genes GRHL3 and PHLDA3 at the transcriptome, proteome, and promoter methylation levels. Based on the results, we hope that further studies will confirm the potential of GRHL3 and PHLDA3 as prognostic biomarkers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12070-024-05057-0.