This paper reports on the stiffness characterization of microfabricated tri-layer conducting polymer (PPy) actuators. The rectangular, polypyrrole microactuators, which could operate both in aqueous and non-aqueous media, were fabricated using an excimer laser ablation technique that provided high throughput production and did not require cleanroom facilities. The microactuators were fixed at one end with electrical contacts and the other was end free to act as an electroactive microcantilever beam. An atomic force microscope (AFM) was used to measure the microactuator deflection under a range of normal forces applied by the AFM cantilever. A modified reference spring constant calibration method was employed to determine the stiffness constants of the microactuators. The stiffness of the microactuators in the electroactive (electrically stimulated) and passive state (no stimulation) were evaluated separately and compared. In doing so, the study presents results leading to the stiffness characterization of the first air-operated polymer microactuators and implementation of a simple, reliable and effective method for directly measuring the spring constant of polymer microactuators. This method is an alternative to the use of mechanical modeling methods, which can be difficult to implement for multi-layer (composite) polymer actuators. Importantly, our results highlight several requirements for using the reference spring method to accurately determine stiffness values of any microcantilever generally fabricated from soft, deformable materials.