In this chapter, we introduce the usage of pHluorin-mKate2-human LC3 for monitoring autophagy. Using EGFP and RFP, tandem fluorescent protein-tagged LC3 has been generated for monitoring autophagic structures. A critical point for this purpose is the sensitivity of the green fluorescent protein to acidic pH. A super-ecliptic pHluorin is most sensitive to acidic pH among EGFP, mWasabi, and pHluorin, indicating pHluorin is most suitable for monitoring autophagic structures. During autophagy, green-positive and red-positive fluorescent puncta of pHluorin-mKate2-human LC3 indicate signals of preautophagosomes and autophagosomes. After fusion of autophagosomes with lysosomes to form autolysosomes, green fluorescence of this intraautophagosomal protein is abolished according to acidification of autolysosomes. Therefore, these green-negative and red-positive fluorescent puncta reflect autolysosomes, in which intraluminal proteins are finally degraded by lysosomal proteases. To monitor autophagic flux, the accumulation of its green-negative and red-positive fluorescent puncta is monitored by inhibiting major lysosomal proteases, cathepsins. In addition, a mutant pHluorin-mKate2-human LC3△G is also introduced as a negative control probe.