Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody–antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4–10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.