Clinicians need a better way to accurately monitor the concentration of antimicrobials in patient samples. In this report, we describe a novel, low-sample-volume method to monitor the azole-class antifungal drug posaconazole, as well as certain other long-chain azole-class antifungal drugs in human serum samples. Posaconazole represents an important target for therapeutic drug monitoring (TDM) due to its widespread use in treating invasive fungal infections and well-recognized variability of pharmacokinetics. The current "gold standard" requires trough and peak monitoring through high-pressure liquid chromatography (HPLC) or liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Other methods include bioassays that use highly susceptible strains of fungi in culture plates or 96-well formats to monitor concentrations. Currently, no method exists that is both highly accurate in detecting free drug concentrations and is also rapid. Herein, we describe a new method using reduced graphene oxide (rGO) and a fluorescently labeled aptamer, which can accurately assess clinically relevant concentrations of posaconazole and other long-chain azole-class drugs in little more than 1 h in a total volume of 100 µl.IMPORTANCE This work describes an effective assay for TDM of long-chain azole-class antifungal drugs that can be used in diluted human serum samples. This assay will provide a quick, cost-effective method for monitoring concentrations of drugs such as posaconazole that exhibit well-documented pharmacokinetic variability. Our rGO-aptamer assay has the potential to improve health care for those struggling to treat fungal infections in rural or resource-limited setting.