jats:titleAbstract</jats:title>jats:pA previous preliminary study of 15th-century Venetian manuscript fragments by the Master of the Murano Gradual identified the presence of cobalt in many ultramarine blue areas, suggesting the presence of smalt. This would represent an early use of this glassy pigment in Venetian illuminated manuscripts. Whereas sampling has been used to identify smalt in 15th century paintings, only non-invasive methods can be used on manuscripts due to their small size and fragile nature. Here we investigated four non-invasive analysis techniques to identify small amounts of smalt in the presence of ultramarine, including single-point and scanning XRF spectroscopy, UV–vis-NIR-SWIR reflectance spectroscopy (FORS), Raman spectroscopy, and external reflection FT-IR spectroscopy. This was done by studying paint mock-ups of ultramarine and smalt mixtures with and without the presence of a white pigment on parchment. The results showed molecular spectroscopy techniques (reflectance, Raman, and FTIR) require at least ~ 30–40% smalt by percent mass when in the presence of ultramarine in order to detect its presence, whereas elemental XRF spectroscopy can detect cobalt (and thus infer the presence of smalt) at the ~ 1% level. To further explore the inference of smalt by XRF, additional XRF analysis was conducted to specifically look for elements associated with cobalt minerals (i.e. nickel, arsenic, bismuth, etc.). High spatial resolution XRF scanning (60–100 μm X-ray spot size) was used to look for cobalt in smalt particles which are typically larger than those of ultramarine. These two XRF analysis approaches worked well with the mock-up paint samples, and were subsequently applied to the manuscripts for which molecular spectroscopy methods yielded no unambiguous evidence for smalt. The data underscore the challenges of conclusively identifying smalt in complex paint systems when samples are not available, but do suggest that the Master of the Murano illuminated manuscript fragments contain smalt, but perhaps not in a form or amount researchers are used to seeing in paintings.</jats:p>