In this study, dissolving polymeric microneedle (MN) patches composed of gelatin and sodium carboxymethyl cellulose (CMC) were used to localize insulin. Their in vitro skin insertion capabilities were determined using tissue-marking dye to stain the skin after patches removal. Scanning electron microscopy (SEM) was used to determine changes in the MNs over time, and optical coherence tomography (OCT) was used to monitor their real-time penetration depth. Confocal microscopy images revealed that rhodamine 6G gradually diffuses from the puncture sites to deeper dermal tissue. Using an in vivo imaging system (IVIS), skin areas that received FITC-insulin-loaded MNs were found to present strong fluorescent signals that greatly decreased 1 h after application. Results show that dissolving MNs rapidly release FITC-insulin, and it then gradually diffuses into the skin. This study verifies that using a gelatin/CMC MN patch for insulin delivery achieves satisfactory relative bioavailability compared to a traditional hypodermic injection and can be a promising delivery device for poorly permeable protein drugs such as those used to treat diabetes. Insertion tests on human cadaveric skin demonstrate that dissolving MNs could serve as efficient devices for transdermal drug delivery in clinical practice and that the volar aspect of forearm skin is the ideal location for their applications.