In this study, we present a method for ion-associated liquid phase (IALP) separation and concentration of analytes from an aqueous matrix into an IALP formed in situ by the charge neutralization reaction of organic cations and anions, without centrifugation. The effects of various factors on the extraction efficiency and other parameters are investigated, whereas no instrumental stirring, such as vortexing or ultrasonics, is required because the solvent (IALP) is formed in situ. The organic cation and anion used are ethylhexyloxypropylammonium and dodecyl sulfate, respectively. The developed in situ IALP microextraction method for phase separation without centrifugation is tested using the thymol blue dye and several endocrine disruptors. The tested endocrine disruptors (bisphenol A, 17β-estradiol, 17α-ethinylestradiol, and estrone) are analyzed via high-performance liquid chromatography/fluorescence detection, with respective detection limits of 0.02, 0.02, 0.02, and 0.4 μg L−1, and the corresponding enrichment factor ranging from 47 to 71. This IALP microextraction method can be used to separate and concentrate environmental water samples of different matrices. The employed IALP is fast and easy to use, enables an approximately 100-fold analyte concentration, and has a high affinity for estrogens, thus holding promise for the separation, concentration, and quantitation of diverse trace analytes.