Fluorescein is a pharmaceutical compound and also a dye. The sodium salt of fluorescein, is used extensively as a diagnostic tool in the field of ophthalmology and is used in the diagnosis of corneal abrasions, corneal ulcers, and herpetic corneal infections. In addition, fluorescein can be considered as an environmental pollutant. For example, fluorescein dye solutions are used as an aid to leak detection during hydrostatic testing of subsea oil and gas pipelines and other subsea infrastructure. In this regard, the development of sensitive, selective and green analytical methods for the enrichment, determination and quantification of fluorescein dye is extremely necessary and important from the environmental and toxicological applications point of view. Dispersive liquid-liquid microextraction (DLLME) has become one of the most interesting sample preparation techniques developed in recent years and has been applied to aqueous samples for determining specific groups of pollutants [1,2]. However, to the best of our knowledge, the application of this technique for the extraction/enrichment of fluorescein in biologic and environmental samples followed by HPLC-UV-vis detection has not been reported so far. In this article, a novel extraction methodology based on dispersive liquid-liquid microextraction coupled with high-performance liquid chromatography-UV-vis detection was exploited for sample preparation and subsequent determination of fluorescein in biologic and environmental samples. Extraction parameters such as ionic strength, pH, mixing assistance, the time required, and different variants of emulsion formation were studied and optimized. The HPLC system consisted of the C18 column (250 mm×4.6 mm, 5 μm) with a mobile phase containing acetonitrile: water (40:60 v/v, pH adjusted to 4.0 with ortho-phosphoric acid) at a flow rate of 1.0 mL min-1 and ambient temperature. UV-vis detection at 494 nm was employed to monitor the analytes. The standard curve was linear wit
