In this study, a novel approach for effective liquid-liquid microextraction based on deep eutectic solvent (DES) decomposition was suggested for the first time. It was established that DESs synthesized from tetrabutylammonium bromide and long-chain alcohols decomposed in aqueous phase resulting in in situ dispersion of organic phase and extraction of hydrophobic analyte(s). It this process long-chain alcohol acted as an extraction solvent and tetrabutylammonium bromide acted as a dispersive agent and promoted mass transfer between aqueous and organic phases as a salting out agent. Phenomenon of DES decomposition was studied in detail and applied for separation and preconcentration in chemical analysis for the first time. The developed approach was applied for 17β-estradiol microextraction from transdermal gel samples as a proof-of-concept example. The results showed that the in situ dispersed organic phase obtained can provide efficient extraction of 17β-estradiol with good extraction recovery (95 ± 5%) and excellent reproducibility (6%). The reported approach proves to be fast, simple, and inexpensive.