Abstract Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The phasor approach to FLIM data is a fit-free analysis and is increasingly used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available thus limiting the widespread use of phasor analysis in biomedical research. Here we present (F)luorescence (L)ifetime (U)l(t)imate (E)xplorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of FLIM analysis, such as calibrating the FLIM data, performing interactive exploration of the phasor plot with cursors, displaying the phasor plot and the FLIM images with different lifetime contrasts and calculating the relative concentration of molecular species. The final edited datasets after applying the desired filters and thresholds can be exported for further user specific analysis. FLUTE was tested using several FLIM datasets including autofluorescence of Zebrafish embryos, cells in vitro and intact live tissues. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets and accelerates FLIM analysis for non-specialized labs. Impact statement This work introduces the first open-source graphical user interface (GUI) for phasor analysis of Fluorescence Lifetime Microscopy (FLIM) data. Phasor analysis is increasingly used for FLIM data analysis in biomedical research as it reduces the complexity of the analysis and provides a powerful visualization of the data content and optimization of data handling with respect to multiexponential fitting. However, the development of quantitative FLIM applications in the life sciences has been until now hampered by the lack of an open source and user-friendly graphical user interface. Here we introduce FLUTE that expands some possibilities of phasor FLIM image processing, accelerates the whole FLIM analysis and simplifies the visualization and the analysis of FLIM data, thus making phasor analysis possible for a broader base of researchers. FLUTE will be of interest to researchers with interests ranging from physics to biology and will facilitate research in several biomedical fields.