The content of pungent compounds in chili peppers does not always directly correlate with the perceived intensity of spiciness in humans. This discrepancy highlights the complex nature of spiciness perception and underscores the need for a comprehensive approach that combines both instrumental analysis and sensory evaluation. We combined instrumental analysis (u-HPLC) with sensory evaluation by descriptive analysis with trained panelists. To examine how the food matrix affects spiciness perception, we compared water and oil extracts of chili pepper powder. Our findings revealed a notable matrix effect: oil extracts were perceived as less spicy than water extracts within the same capsaicinoid content. This phenomenon could be attributed to several factors, including a coating effect where oil might inhibit direct contact between capsaicin and transient receptor potential vanilloid 1 (TRPV1) receptors and flavor interference from other compounds extracted by oil. This suggests that spiciness perception is not solely determined by capsaicinoid content but also by the complex interactions between the capsaicinoid and the food matrix.