Low temperature plasma (LTP) ionization is an ambient plasma ionization method that permits the direct mass analysis of samples in their native atmospheric environment with little or no sample preparation. In this work, the low temperature plasma probe is used in the direct and rapid mass spectrometric analysis of aqueous phase samples including biofluids (saliva, urine, and hair extract). A detailed trace qualitative examination of 14 drugs of abuse has been performed. The relative standard deviation on average was ∼16% for the LTP analysis of the drugs of abuse standards. Eleven of the fourteen drugs of abuse were detected in the low ng mL⁻¹ (3 pg absolute detection) to the mid µg mL⁻¹ (∼30 ng absolute detection) concentration range. One drug, cannabidiol, could not be detected until supplemental heating of the substrate was incorporated into the experimental protocol. The addition of supplemental heating improved the detection limits by at least an order of magnitude to ∼0.5 ng mL⁻¹ to 0.5 µg mL⁻¹ (1.5 pg–1.5 ng absolute) for twelve of the fourteen drugs of abuse, so extending the linear dynamic range which for most analytes was four orders of magnitude. Quantitative capabilities were evaluated using the particular example of benzoylecgonine in urine by employing a deuterated internal standard. Matrix effects observed during the analysis of the drugs in complex biological fluids are also discussed. In addition, low temperature plasma ionization was applied to the examination of real (not spiked) biological samples and these results were confirmed using standard LC/MS methodology. The main advantages observed for this ambient desorption/ionization technique include the capabilities for direct analysis of liquid surfaces for in situ detection and the remarkable sensitivity in the examination of the drugs of abuse investigated here. The disadvantages of the method include the modest quantitative accuracy making LTP most useful as a rapid but semi-quantitative screening method.