Photoactivated drug delivery systems using gold nanoparticles provide the promise of spatiotemporal control of delivery that is crucial for applications ranging from regenerative medicine to cancer therapy. In this study, we use second harmonic generation (SHG) spectroscopy to monitor the light-activated controlled release of oligonudeotides from the surface of colloidal gold nanoparticles. MicroRNA is functionalized to spherical gold nanoparticles using a nitrobenzyl linker that undergoes photodeaving upon ultraviolet irradiation. The SHG signal generated from the colloidal nanoparticle sample is shown to be a sensitive probe for monitoring the photodeaving dynamics in real time. The photodeaving irradiation wavelength is scanned to show maximum efficiency on resonance at 365 nm, and the kinetics are investigated at varying irradiation powers to demonstrate that the nitrobenzyl photodeaving is a one-photon process. Additional characterization methods including electrophoretic mobility measurements, extinction spectroscopy, and fluorimetry are used to verify the SHG results, leading to a better understanding of the photodeaving dynamics for this model oligonudeotide therapeutic delivery system.