The assessment of the structural condition of cultural heritage objects is important for conservation interventions and their long-term preservation. This investigation concerns The Night Watch (1642), a large-format 17th-century canvas painting by Rembrandt van Rijn that is on display in the Rijksmuseum, Amsterdam. This painting, which has a complex treatment history, has various damaged areas and has undergone three wax-resin relinings. In 1975 the canvas was slashed twelve times with a serrated dinner knife, including several long slashes in the area of Captain Frans Banninck Cocq’s breeches. In 2021, prior to a proposed new structural intervention involving retensioning of the canvas, it was important to evaluate the structural condition of the repaired slashes and of another repair, specifically an old canvas insert in the drum. For this, an in-situ inspection was carried out in the Rijksmuseum as a part of Operation Nightwatch. 3D shearography instrument with thermal loading was used to inspect these two areas of interest on the reverse of The Night Watch. The results showed that the out-of-plane strain in the breeches does not show any large deviations, which alleviated conservators’ concerns about the adhesion of the lining canvas and stability of previous repairs in this region. The patch in the drum showed higher out-of-plane strain variations. This was explained by the lower quality of the patched canvas compared to the repaired slashes in the breeches of Banninck Cocq. Overall, 3D shearography provided valuable inspection results for assurances regarding the structural integrity of the 1975 repairs and the wax-resin lining in The Night Watch, reducing the risks and providing the confidence to proceed with the planned retensioning of the canvas.