A simple, rapid, and sensitive method for the detection of posaconazole using dispersive liquid-liquid microextraction (DLLME) coupled to surface-assisted laser desorption/ionization mass spectrometric detection (SALDI/MS) was developed. After the DLLME, posaconazole was detected using SALDI/MS with colloidal gold and α-cyano-4-hydroxycinnamic acid (CHCA) as the co-matrix. Under optimal extraction and detection conditions, the calibration curve, which ranged from 1.0 to 100.0 nM for posaconazole, was observed to be linear. The limit of detection (LOD) at a signal-to-noise ratio of 3 was 0.3 nM for posaconazole. This novel method was successfully applied to the determination of posaconazole in human urine samples.