A natural deep eutectic solvent (NaDES) composed of levulinic acid and glucose using a molar ratio of 5:1 (molHBA:molHBD) and 20% of water (w/w) (LeG_5_20) was found as a great alternative to the commonly used organic solvents for the extraction of hydroxynaphthoquinone enantiomers (HNQs) from Alkanna tinctoria roots. In the present work, a comparative investigation of recovery methods for HNQs, such as solid-phase extraction, macroporous resin, and water as anti-solvent, was performed to face the main disadvantage of NaDES: inability to be evaporated. The highest recovery of HNQs was recorded using the solid-phase extraction on a reversed-phase C8 cartridge with a total hydroxynaphthoquinone content (TNC) of 46.79 ± 0.952 mg/g of dry weight (DW). Besides, a great recovery of HNQs was also reported for the macroporous resin Amberlite XAD 4 with a TNC value of 37.21 ± 1.789 mg/g DW while the precipitation of HNQs by using water as an anti-solvent (1:5, v/v) offered a TNC value of 28.68 ± 0.023 mg/g DW. The macroporous resin Amberlite XAD also showed a great potential for larger scale applications. In fact, the developed scale-up process, involving Amberlite XAD 4, showed a great recovery efficiency for HNQs (34.126 ± 1.093 mg/g DW), an acceptable robustness (RSD <
15%) and the possibility of recy-cling LeG_5_20 with a recovery greater than 50%
therefore, an excellent green alternative extrac-tion procedure for HNQs.