This paper presents the design and validation of an electrochemical sensor for the detection of octopamine (OA) in vitro-reared larvae of honey bees (Apis mellifera) using glassy carbon electrodes modified with multi-walled carbon nanotubes and Kolliphor (p188). Octopamine, a key neurohormone involved in the stress response in insects, is studied as an indicator of the physiological and health status of bees under various stress factors. To validate the electrochemical method, a chromatographic approach based on ultra-high-performance liquid chromatography with fast-scanning fluorescence detection was developed, as well as a retro-microextraction system optimized using design of experiment and response surface methodology. The electrochemical method achieved limits of detection and quantification of 10.8 ng mL