Proteins and small molecules from ancient objects and cultural heritage can provide key information and contribute to study the context of objects and artists. However, all present-day protocols and strategies for the analysis of ancient samples are often invasive and require microsampling. Here, we present a new method for the noninvasive analysis of proteins and small molecules: the technique uses a special ethyl-vinyl acetate film functionalized with strong cation/anion exchange and C8 resins, for interacting with both proteins and small molecules present on the surface of the objects, followed by LC-MS/MS analysis. The new method was fully validated for the determination of both proteins and small molecules on several types of supports, showing excellent analytical performances such as, for example, R2 of the calibration curve of 0.98 and 0.99 for proteins and small molecules, low but very repeatable recoveries, particularly adequate for investigations on precious ancient samples that must not be altered by the analytical procedure. ESEM images and LED multispectral imaging confirmed that no damages or alterations occurred onto the support surfaces and no residues were left from the extractive film. Finally, the new method was applied for the characterization of the binders of a historical fresco of the XVI century from the Flemish painter Paul Brill and of a recently discovered fresco from Isidoro Bianchi (XVII century). Moreover the method was employed for the identification of the colorant used by Pietro Gallo (XIV century) on a wood panel. The method here reported can be easily applied to any other research on ancient precious objects and cultural heritage, since it does not require microsampling and the proteins/small molecules extraction can be performed directly in situ, leaving the object unchanged and intact.