Solvent-based ambient ionization mass spectrometry (MS) techniques provide a powerful approach for direct chemical analysis and molecular profiling of biological tissues. While molecular profiling of tissues has been widely used for disease diagnosis, little is understood about how the interplay among solvent properties, matrix effects, and ion suppression can influence the detection of biological molecules. Here, we perform a systematic investigation of the extraction processes of lipids using an ambient ionization droplet microsampling platform to investigate how the physicochemical properties of the solvent systems and extraction time influence molecular extraction and detection. Direct molecular profiling and quantitative liquid chromatography-mass spectrometry (LC-MS) of discrete solvent droplets after surface sampling were investigated to provide insights into extraction and ionization mechanisms. The results of this study suggest that intermolecular interactions such as hydrogen bonding play a major role in extraction and detection of lipids using solvent-based ambient ionization techniques. In addition, extraction time was observed to impact the molecular profiles obtained, suggesting optimization of this parameter can be performed to favor detection of specific analytes.