Aim of this study was to demonstrate the influence of different analytical procedures and techniques on the resulting miRNA expression profile in healthy control subjects and tumor patients using the oral squamous cell carcinoma (OSCC) model and to demonstrate the technical and biological reproducibility. Body fluids such as saliva are suitable for non-invasive miRNA analysis because ubiquitously circulating miRNA can be found in them. It was technically possible to distinguish between healthy and diseased samples based on the miRNA expression profile found. Regardless of the methodology used, good technical reproducibility of the results seems to be achievable. On the other hand, biological reproducibility was inadequate, which is why prompt sampling and sequencing is recommended. The data indicate that malignant lesions can be detected using miRNA signatures extracted from saliva. This could stimulate further research to establish standardized protocols and kits for sample collection, miRNA extraction, sequencing and interpretation of results.