A novel analytical method to determine five chromophore-lacking macrolide antibiotics in human urine was established. This method combined  dispersive liquid–liquid microextraction based on the solidification of floating organic droplets (DLLME-SFO) and liquid chromatography with charged aerosol detection (LC-CAD) for the first time. CAD allowed the sensitive detection of macrolide antibiotics lacking chromophores while DLLME-SFO enabled rapid and efficient sample clean-up and preconcentration. Parameters influencing the microextraction efficiency were optimized using one-variable-at-a-time approach. The resulting optimized conditions yielded high enrichment factors in the range of 60–106 and the extraction efficiency was very high as indicated by the measured absolute recovery values close to 100%. The developed method exhibited the limit of detection (LOD) as low as 10 to 40 ng mL−1 and intra-day and inter-day precisions below 8.7% and 12.6%, respectively. The method was compared with the conventional sample preparation methods including the liquid–liquid extraction and organic solvent precipitation methods and it was found that the proposed method was more sensitive, specific,rapid, and environmentally friendly.The current study suggests that the combined use of DLLME-SFO and LC-CAD may be applicable to the analysis of various compounds with poor to no chromophores in complex matrices.