Squamous cell carcinoma is a malignancy of squamous epithelia and affects multiple organs of the body. Investigating the underlying signalling pathways and the molecular mechanisms is essential to gain a profound understanding of the disease pathology, aetiology and to uncover potential drug targets. The deubiquitylating enzyme, USP9X, has been associated with epithelial cancers of breasts, colon, lung, liver, pancreas, mouth and the skin. The current study investigated the functional and molecular role of USP9X in head and neck squamous carcinoma cell lines (HNSCC) and skin keratinocytes. Also, the role of USP9X in DNA damage response of keratinocytes was investigated. USP9X protein expression was altered in four HNSCC cell lines, derived from tongue (SCC15 and CAL27) and pharynx (Fadu and Detroit 562) by treating them with siRNAs (loss of function) or by transfecting them with plasmids (gain of function). CyQuant and Flow cytometry analyses revealed that USP9X promoted proliferation and regulated cell cycle of these cell lines. RT-qPCR analysis of target gene expression revealed that USP9X also regulated Notch activity in these cells. However, at least in one cell line (FaDu), USP9X’s regulation of proliferation did not appear to operate through the Notch pathway but possibly occurs via mTOR pathway. Immunoblot analysis revealed a decrease in downstream targets of mTOR complex 1 namely, total ribosomal protein (S6) and its phosphorylated form (pS6), when USP9X was depleted from FaDu cells. In contrast, in immortalised but non- tumourigenic HaCaT human keratinocytes, USP9X depletion led to an increase in cell proliferation and again, USP9X levels directly correlated with Notch pathway activity.