<p>  </p> <p>Miniature mass spectrometers are in a phase of rapid development due to their potential in offering simple yet powerful solutions for a wide variety of unmet biomedical needs. In particular, the combination of ambient ionization methods with miniature mass spectrometers offers an attractive solution for improving patient outcomes and reducing the healthcare burden on patients as well as clinicians opposed to current methods for disease prognosis and diagnosis. </p> <p>There has been a rapid expansion in the commercial offerings of miniature mass spectrometers from commercial vendors, both large and small, including Purspec, Bayspec, MassTech, Waters, and Advion. Despite the large number of instruments that have been made available and the success of many of these systems with analysis of a broad range of biological matrices, much work remains to perform bioanalysis of complex molecules with concentrations that often lie in the ng/mL-µg/mL range. Miniaturization of mass spectrometers is accompanied by design simplifications in comparison to benchtop instruments, so that sacrifices are often made in terms of performance. The figure of merit that is compromised, of course, depends on the design of the instrument itself. Thus, in addition to a good understanding of the operation of the mass spectrometer, it is crucial that the ionization method for the analyte be chosen judiciously, and that the method is suitably optimized to be able to perform the measurements to obtain high quality data for trace analysis. </p> <p>The custom built Mini-12 miniature MS systems at Purdue University is one such miniature mass spectrometer that can be used for making on-site measurements. In this work, I have described my efforts to perform trace analysis of a range of molecules (tenofovir diphosphate, cabotegravir, rilpivirine, and phosphatidylethanol) relevant to HIV treatment and prevention with the Mini-12 system. Based on the most favorable set of conditions for developing a particular assay, method performance parameters are listed for each of the applications described. In all the above applications, the desired detection limits are met by adopting a broad range of strategies with the focus of keeping the method amenable to use at the point-of-care, i.e., ensuring that sample preparation is completed in <4 minutes. The proof-of-concept results obtained with the custom built Mini-12 mass spectrometer at Purdue University lays the groundwork to (i) encourage the introduction of miniature mass spectrometer-based assays for the molecules listed in a clinical setting and (ii) extend the use of miniature mass spectrometer-based assays for other therapeutic regimens that require longitudinal monitoring.</p>