Application of monoisotopic cationic 109Ag nanoparticles (109AgNPs) obtained by pulsed fiber laser (PFL) 2D galvo‐scanner (GS) laser generated nanomaterial (LGN) for both high resolution laser desorption/ionization mass spectrometry and mass spectrometry imaging of amino acids is presented. Four amino acids, alanine, isoleucine, lysine, and phenylalanine were used as test compounds for quantification with matrix‐assisted laser desorption/ionization mas (MALDI)‐type mass spectrometer. Comparison of commonly made manual measurements with semiautomatic mass spectrometry imaging (MSI) was performed providing very interesting findings. Amino acids were directly tested in 1 000 000‐fold concentration change conditions ranging from 1 mg/ml to 1 ng/ml, which equates to 500 ng to 500 fg of amino acid per measurement spot. Methods were also tested on samples of human blood plasma for quantification of endogenous amino acids.