We present an automated MATLAB-based GUI for the analysis of microfluorimetry data inisolated neuronal cultures. The pipeline integrates signal extraction, quality control,responder classification, and stimulus-evoked quantification into a single workflowcompatible with both single-wavelength and ratiometric dyes. Applied to thermal and drugstimulations, the automated analysis reproduces the classification and quantitative outputsobtained using manual approaches, while substantially reducing analysis time. The GUI wedeveloped provides a practical and reproducible solution for higher throughput [Ca²⁺]ᵢimaging analysis across neuroscience, cell biology, and related fields.