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In this work, a novel procedure based on surfactant-assisted directly suspended droplet microextraction for the determination of tramadol prior to GC with flame ionization detection is proposed. In this technique, a free microdroplet of solvent is transferred to the surface of an immiscible aqueous sample containing Triton X-100 and tramadol while being agitated by a stirring bar placed on the bottom of the sample vial. After the predetermined time, the microdroplet of solvent is withdrawn by a syringe and analyzed. The effective parameters such as the type of organic solvent, extraction time, microdroplet volume, salt content of the donor phase, stirring speed, the source phase pH, concentration of Triton X-100, and extraction temperature were optimized. For this purpose, a multivariate strategy was applied based on an experimental design in order to screen and optimize the significant factors. This method requires minimal sample preparation, analysis time, solvent consumption, and represents significant advantages over customary analytical methods. The linearity ranged from 10 to 2000 μg/L with RSDs (n = 5) of 7.3-10. Preconcentration factors and the LODs were 391-466 and 2.5-6.5 μg/L, respectively. Finally, this method was applied to the analysis of biological samples and satisfactory results were obtained.