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One of the reactive forms of oxygen is hydrogen peroxide (H2O2), which has been investigated as a key component of growth processes and stress responses. Different methods for the determination of H2O2 production by animal and bacterial cells exist; however, its detection in algal cell cultures is more complicated due to the presence of photosynthetic pigments in the cells and the complex structure of cell walls. Considering these issues, a reliable, quick, and simple method for H2O2 detection is needed in phycological research. The aim of this methodological study was to optimize an Amplex UltraRed method for the fluorometric detection of H2O2 produced by microalgae cells, using a wild‐type strain of Chlamydomonas reinhardtii as a model. The results showed that (i) potassium phosphate is the most suitable reaction buffer for this method, (ii) a 560 nm wavelength variant is the most appropriate as the excitation wavelength for fluorescence spectra measurement, (iii) a 50:50 ratio for the reaction mixture to sample was the most suitable, (iv) the fluorescence signal was significantly influenced by the density of the microalgae biomass, and (v) sample fortification with H2O2 allowed for an increase of the method's reliability and repeatability. The proposed protocol of the Amplex UltraRed method for the fluorometric detection of H2O2 produced by microalgae cells can yield a sensitive and accurate determination of the content of the test compound, minimizing measurement errors, eliminating chlorophyll autofluorescence problem, and compensating for the matrix effect. This method can be applied to the study of other microalgae species. [ABSTRACT FROM AUTHOR]