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The method describes pigment analysis from microalgae/phytoplankton extracts using electron-transfer Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (ET MALDI MS). Current microalgae/phytoplankton pigment analysis requires resource- and time-intensive chromatographic methods due to the broad polarity range of the target analytes. On the other hand, traditional MALDI MS chlorophyll analysis, using proton-transfer matrices such as 2,5-dihydroxybenzoic acid (DHB) or α-cyano-4-hydroxycinnamic acid (CHCA), results in central metal loss and phytol–ester cleavage. ET MALDI MS is an alternative for the rapid screening and detection of pigments in microalgae extracts. • MALDI matrices with ionization energies above 8.0 eV guarantee electron-transfer processes from photosynthetic and photoprotective pigments whose ionization energies lay below 7.5 eV. • ET MALDI MS pigment analysis agrees with data gathered from conventional chromatographic techniques (HPLC) and optical microscopy for pigment extracts from C. vulgaris cultures and freshwater phytoplankton samples. • The ET MALDI MS method allows fast and reliable detection of pigments in microalgae cultures and freshwater phytoplankton samples.