The goal of this study is to develop a non-invasive, accurate, and cost-effective saliva-based test for early detection of head and neck squamous cell carcinoma (HNSCC). To this end, we have developed a panel of seven biomarkers through screening of methylated microRNA loci (i.e. 9-1, 124-1, 124-2, 124-3, 129-2, 137 and 148) for HNSCC, and hereafter referred to as HNKlear. HNKlear demonstrated 92.1% sensitivity and 97.8% specificity in 189 HNSCC and 92 control tissue samples and 84.9% sensitivity and 95.4% in 86 pre-treatment HNSCC and 108 control saliva samples. HNKlear is able to detect 93.5% T1 tumor and 91.2% stage I tumor in tissue, and 85.0% or 80.0% of HNSCC saliva when tumor is at T1 or stage I, respectively. To further validate and develop HNKlear as a robust clinical test for HNSCC, we have validated HNKlear both analytically using the CLIA lab setting, and clinically using the external cohorts with support of the NCI UH2/UH3 funding on “Assay validation of high-quality markers for clinical studies in cancer”. In analytical validation, we have determined the Limit of Detection by spiking both HPV+ HNSCC cell line, UMSCC47, and HPV- HNSCC cell line, FaDu into a pool of saliva matrix. The non-HNSCC oral epithelial cell OKF6 was used to determine the specificity of HNKlear. The stability of saliva collected in the Saliva Collection Device (DNA Genotek) was determined when samples were stored at different temperatures for up to one week. The precision and reproducibility of HNKlear were assessed by intra-run, inter-run, different machines, days and operators. The potential interfering substances for saliva, such as mucin, nicotine, toothpaste, nasal spray etc. were also examined. In clinical validation of the external cohorts, we have performed HNKlear test on 231 HNSCC cases and 182 controls using the previously established algorithm and cut-off. The sensitivity and specificity of HNKlear in external cohorts were 83.5% and 95.3% respectively. In summary, the saliva liquid-biopsy test, HNKlear has been validated and has potential to develop as a robust clinical test for HNSCC.