Various intensiometric biosensors have emerged to date, capable of monitoring concentration changes in intracellular signaling molecules as fluorescence intensity changes. However, these biosensors frequently encounter difficulties in quantitative analysis due to their inherent problem of intensity-based analysis. To overcome this limitation, fluorescence lifetime imaging microscopy (FLIM) has garnered attention as a more quantitative approach based on robust physicochemical parameters. Recently, we have developed several FLIM-based biosensors, using both genetically encoded biosensors and small chemical probes. For instance, we designed biosensors by linking GFP variants with binding domains for the target analyte through optimal peptide linkers. Consequently, we successfully generated FLIM biosensors for ATP and Ca²⁺. Notably, the Ca²⁺ sensor proved effective for endoplasmic reticulum and mitochondria with higher concentrations compared to the cytoplasm. Conversely, we also produced small chemical FLIM probes to detect intracellular temperature at organelles in thermogenic skeletal muscles. Since elements like ATP, Ca²⁺, and intracellular temperature are critical for skeletal muscle homeostasis, in this talk, we demonstrate the potential of these tools for skeletal muscle studies.