A novel molecularly imprinted polymer nanoparticle-based assay (MINA) performed in magnetic microplates was developed as an improved high-quality alternative to existing antibody-based immunoassays. MINA is a generic technology that can be adapted for biomarker detection in biological samples. Herein, we demonstrate the applicability of the MINA assay for the detection of leukotrienes and insulin in biological samples. MINA, used in a competition format, has allowed the detection of LTE4 in urine in a concentration range from 0.45 to 364 pM, with a LOD of 0.73 pM. MINA, used in a competition format, has allowed the detection of insulin in plasma in a concentration range from 25 to 2500 pM, with a LOD of 27 pM. This assay has shown comparable performance for LTE4 and insulin detection to existing chromatographic techniques (LC-MS/MS) and immunoassays in clinically relevant concentrations. The main advantages of this assay are the efficient and low cost fabrication, preparation of synthetic binders without the use of animals, and fewer steps used in the assay protocol as compared to traditional immunoassays.