During a routine condition survey in 2007 at the Georgia O’Keeffe Museum in Santa Fe, micro-protrusions were found scattered across the surfaces of the artist’s oil paintings produced between 1920 and 1950. In many of her works, including Pedernal (1941) and A Man from the Desert (1941), lead soaps were found aggregated at the painting surface, forcing the surrounding paint to deform into pin-sized protrusions. The structure and composition of the protrusions was analyzed to determine why they formed. Microsamples were removed from four of O’Keeffe’s paintings and investigated using a combination of SEM–EDX, GC–MS, and FT-IR. The combined GC–MS and FT-IR results indicate that the protrusions contain primarily lead carboxylates. The results obtained for a series of ground samples removed from four different paintings were then compared with samples collected from a commercially pre-primed canvas roll found in the artist’s house in Ghost Ranch, NM. We identified two different types of canvases: i) a commercially pre-primed canvas and ii) artist primed canvas. This commercially pre-primed canvas roll has a similar morphology to the ground layers in her paintings that contain protrusions additionally these grounds were found to contain an excess of free fatty acids and metal carboxylates (lead soaps) based on a comparison to canvases the artist primed herself. Based on the analytical results from the four paintings and the commercial pre-primed canvas, we concluded that the artist’s use of this particular commercially pre-primed canvas between 1920 and 1950 play an important role in the observed micro-protrusions.