In the microscopic analysis of living organisms such as cells and tissues, autofluorescence is a drawback for overlapping photoluminescence signals. In particular, short-lived autofluorescence overlaps with weak luminescence signals, which limits reliable signal detection. To reduce this interference, selective detection of luminescence signals using time-gated analysis provides an effective strategy.We developed and applied a time-gated in-situ microscopic and spectroscopic analysis based on the Accumulated In-situ Spectral Information Analysis for phosphorescence signal detection. By combining time-gated microscopy with in-situ spectroscopy, the system enables the simultaneous measurement of microscopic images and emission spectra at the same location. In addition, signal accumulation improves the SBR and SNR. Using this method, autofluorescence signals are selectively excluded, allowing clear imaging and spectral analysis of weak luminescence signals.