We have prepared a microfluidic paper-based analytical device for the determination of cysteine and homocysteine based on 1,5-diphenylcarbazide-capped silver nanoparticles. The development of µPAD was intended to identify and quantify levels of cysteine and homocysteine. The proposed µPAD allowed to detection of cysteine and homocysteine by a colorimetric reaction based on modified silver nanoparticles. The color of modified AgNPs in the test zone immediately changes after addition of cysteine and homocysteine. Based on this change quantification of these two amino acids was achieved with RGB color model using ImageJ software. In optimized conditions, the proposed device was enabled to determination of cysteine in the 0.20-20.0 μM concentration range with 0.16 µM of limit of detection (LOD). Also, the detection limit of homocysteine was calculated to be 0.25 µM with a linear range of 0.50-20.0 μM. In this work; we focused on µPADs usage in analysis of a series of human urine samples.