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DBS has drawn increasing attention as a versatile means of sample collection to support pharmacokinetics and toxicokinetics studies for its distinctive advantages. A small volume of blood, typically 10-20 µL, is spotted onto the DBS card. The DBS sample size available for LC-MS/MS analysis is usually 20-100 times less than a plasma assay. It is vital to increase LC-MS/MS sensitivity to accommodate DBS bioanalytical studies. Strategies to significantly enhance assay sensitivities (>20-fold) were developed via efficient utilization of sample extracts. For nonpolar compounds, e.g., lansoprazole, trap columns based on reversed-phase materials, such as C8 and C18, are employed for sample enrichment prior to analytical separations on a C18 column. For polar compounds like clonidine, a twodimensional separation (2D-LC) strategy was developed. The first dimension is based on a strong cation exchange column, which concentrates clonidine while other un-retained compounds are washed. The salt concentration is then increased to elute clonidine to a C18 column—the 2nd dimension separation, where desalting and analytical separation takes place. For both strategies, the online trapping was demonstrated with up to 100-µL injections while good chromatographic peaks are maintained. Compared to a normal injection of < 5µL, the assay sensitivity can be enhanced by at least 20-fold.