<p>Understanding the neural mechanisms underlying sensory perception remains a fundamental challenge in neuroscience. In vivo two-photon calcium imaging in behaving mice has become a widely used approach to dissect cortical dynamics during perceptual decision-making. In this protocol, we present a standardized and reproducible experimental pipeline for investigating the neural basis of visual perception at cellular resolution. After being surgically prepared, the mice were trained in a step-by-step method and reliably learned the orientation-based visual discrimination task at full contrast, achieving high performance within days and maintaining stable proficiency across sessions. Combining viral-mediated expression of genetically encoded calcium indicators with two-photon imaging, deep-layer neuronal activity in the primary visual cortex was recorded while mice were performing the multi-contrast discrimination task. The imaging data revealed diverse neuronal response profiles, providing insights into the cortical representations supporting visual perception. Our protocol provides detailed information on surgical preparation, behavioral training and data acquisition, offering educational and practical guidance for investigating sensory processing and perceptual decision-making in the mammalian cortex.</p>