There is growing demand for rapid, nondestructive detection of trace-level bioactive molecules including medicines, toxins, biomolecules, and single cells, in a variety of disciplines. In recent years, surface-enhanced Raman scattering has been increasingly applied for such purposes, and this area of research is rapidly growing. Of particular interest is the detection of such compounds in dried saliva spots (DSS) and dried blood spots (DBS), often in medical scenarios, such as therapeutic drug monitoring (TDM) and disease diagnosis. Such samples are usually analyzed using hyphenated chromatography techniques, which are costly and time consuming. Here we present for the first time a surface-enhanced Raman spectroscopy protocol for the detection of the common antidepressant amitriptyline (AMT) on DBS and DSS using a test substrate modified with silver nanoparticles. The validated protocol is rapid and non-destructive, with a detection limit of 95 ppb, and linear range between 100 ppb and 1.75 ppm on the SERS substrate, which covers the therapeutic window of AMT in biological fluids.