In this study, a simple method using microextraction by packed sorbent and high-performance liquid chromatography with ultraviolet detection for simultaneous determination of chlorpropamide, gliclazide and glimepiride in human plasma was developed and validated. A fractional factorial design and a complete factorial design were applied to evaluate the parameters which could affect the extraction and desorption steps, respectively. All parameters in the extraction step (pH, sample volume, sample dilution and number of aspiration/ejection cycles) and in the desorption step (percentage of acetonitrile in the elution solvent and number of aspirations of elution solvent through the device) were statistically significant (p>0.05) when recovery was used as response. The developed method allowed the use of small volumes of sample and solvents and rapid separation by using a fused core column (only 2.2min were needed). This method was fully validated showing selectivity, precision, accuracy and linearity over the range 1.0-50.0μgmL(-1) for chlorpropamide, 1.0-10.0μgmL(-1) for gliclazide and 0.1-1.0μgmL(-1) for glimepiride. Finally, the validated method was applied in the analysis of samples from volunteers containing the three tested analytes.