It this paper, the comprehensive design process was carried out to fabricate selective, molecularly imprinted polymer (MIP). The material was used as a sorbent in the optimized analytical method, aimed at verifying the hypothesis that the conditions of an analytical process could convert 4-hydroxyphenylacetic acid to 4-hydro-3-nitrophenylacetic acid, comprising a series of novel studies. The design stage consisted of the analysis of specificity of the MIP, synthesized from the cross-linker and used five various functional monomers independently, in the presence of four different templates. The MIP from 1-vinylimidazole, imprinted by 4-hydroxyphenylacetic acid, revealed the highest specificity in relation to 4-hydro-3-nitrophenylacetic acid, with an affinity factor equal to 3, and the highest selectivity from a group of structurally similar and biologically important biomolecules. The theoretical analysis revealed that electrostatic interaction between the analyte and the polymer matrix enhanced selectivity. The physicochemical characterization showed the specific surface area of the MIP as being equal to 368.6 m(2) g(-1), and the presence of nitrogen atoms at the level of 6.80% wt., confirming the monomer residue in the material structure. The MIP was applied in the solid phase extraction protocol, allowing for the analysis of 4-hydroxy-3-nitrophenylacetic acid in a human urine sample. Finally, the conversion of 4-hydroxyphenylacetic acid in human urine in nitrate and nitrite salts at low pH conditions revealed an almost twofold increase in 4-hydro-3-nitrophenylacetic acid to 775 +/- 81 ng L-1. The results also confirmed the applicability of the new MIP sorbent for the purpose of analysis of low levels of analyte, present in the complex sample.