Background: Every minor and major surgical procedure requires anesthesia to relieve pain during surgery. The neuro-biomolecular mechanism of pain relief in hypnoanesthesia remains uncomprehended. Objective: This study aims to observe the effect of hypnoanesthesia on several neurotransmitters, including serotonin, glutamic acid, and substance P, which play a role in the mechanism of pain. Methods: The study subjects included patients with benign soft tissue tumors consisting of 40 people who were divided into two groups, namely treatment and control groups. Minor surgery with hypnoanesthesia was performed in the treatment group, whereas in the control group, minor surgery was performed with 2% lidocaine local anesthetic. Pain in both groups was measured by FPS (Face Pain Scale) and monitored by a vital sign monitor. The processes of the study were recorded with a camcorder. Changes in serum levels of excitatory (glutamic acid and substance P) and inhibitory (serotonin) neurotransmitters before and after the intervention were analyzed using ELISA (Enzym-Link Immunosorbent Assay) in both groups. Results: The control and treatment groups had the same end result, which was pain relief. The results of regression and ANOVA analysis indicated that serotonin simultaneously had a significant effect on substance P at 98.4% and glutamic acid at 98.2%. Conclusion: There was no statistically significant change in serotonin levels before and after hypnoanesthesia. Serotonin, as an inhibitory neurotransmitter, simultaneously has a significant effect on both excitatory neurotransmitters, namely Glutamate Acid and Substance P, in the mechanism of nociceptive pain relief with hypnosis.