Singlet oxygen, a harmful reactive oxygen species, can be quantified with the substance 2,2,6,6-tetramethylpiperidine (TEMP) that reacts with singlet oxygen, forming a stable nitroxyl radical (TEMPO). TEMPO has earlier been quantified with electron paramagnetic resonance (EPR) spectroscopy. In the present study, we designed an ultra-high-performance liquid chromatographic – tandem mass spectrometric (UHPLC-ESI-MS/MS) quantification method for TEMPO and showed that the method based on multiple reaction monitoring (MRM) can be used for the measurements of singlet oxygen from both non-biological and biological samples. Results obtained with both UHPLC-ESI-MS/MS and EPR methods suggest that plant thylakoid membranes produce 3.7 x 10-7 molecules of singlet oxygen per chlorophyll molecule in a second when illuminated with the photosynthetic photon flux density of 2000 μmol m-2s-1.This article is protected by copyright. All rights reserved.