To overcome some drawbacks of liquid phase microextraction methods, in this study a novel in-tube solid phase extraction coupled with liquid-liquid-liquid extraction (in tube SPE-LLLE) was developed for extraction and determination of naproxen (NAP) as a template drug in plasma sample. In-tube SPE-LLLE is an extraction technique, in which NAP is extracted from the aqueous and plasma samples directly to inner coated surface of a glassy column by polypyrrole that is inoculated with n-octanol. The analyte can be extracted by both adsorption and absorption mechanisms. Using in-tube SPE-LLLE-HPLC-UV, the calibration curve of NAP in plasma was linear in the range from 0.9–450 ng mL−1 with correlation coefficients above 0.9973 and detection limits (S/N = 3) of 0.54 ng mL−1. The relative standard deviations (n = 5) for recovery of 100 ng mL−1of NAP from 1.0 mL of buffer and plasma samples were 3.2 and 5.6%, respectively. This method was successfully applied for analysis of plasma of three patients administrated NAP.