Peripheral sensitization, decrease in pain threshold in sensory neurons, can cause chronic pain. Little is known about how peripheral sensitization led to chronic pain. Here, we aimed to develop a method for evaluating peripheral sensitization in cultured sensory neurons with electrical recording. Sensory neurons from rat dorsal root ganglion were cultured on high‐density microelectrode arrays (HD‐MEA), and their activity was evoked by capsaicin stimulation with or without substance P. Fluorescent imaging‐based electrode selection effectively selected five times as many capsaicin‐sensitive sensory neurons as the existing method. Peripheral sensitization by substance P was detected from 31.9% of selected sensory neurons, and majority of these neurons co‐expressed capsaicin and substance P receptors. These results indicate that our method is suitable for evaluating peripheral sensitization by substance P in cultured sensory neurons. [ABSTRACT FROM AUTHOR]