The in situ analysis of cellular functional molecules has recently attracted increasing attention because it can provide valuable information for revealing the complex mechanisms of biological events. To achieve in situ cytosensing with high performance, one needs to tailor each of the structural units, including the recognition module, signal transduction module and signal output module, elaborately and integrate them into a system rationally. Several key aspects, such as recognition/binding specificity, signal generation pathway, signal amplification and multi-channel analysis capability, should be taken into consideration when designing a competent system. This chapter describes the principles for addressing these issues and provides a number of representative methods that display superior performance.