Non-invasive matrices such as tears, sweat, saliva, and milk offer an enticing alternative to traditional sampling from blood, serum, and plasma. Collection of these matrices is simpler and more affordable than venipuncture. It is also much less unpleasant for patients and study subjects allowing for more frequent sampling, higher compliance, and removal of fear of needles as a barrier to clinical trial recruitment. However, bioanalysis of these matrices is not without challenges. Sample volumes can be very low, the more complex matrices such as milk may require extensive sample preparation, and the use of the matrix must be biologically relevant. Using the example of a method we developed at Alturas Analytics Inc. to measure Tobramycin from human tears, we will discuss practical applications of microsampling of non-invasive matrices for bioanalysis, and improvements offered by emerging technologies.