This paper presents the pigment characterization in six impressionist and post-impressionist paintings by three leading Puerto Rican artists: Francisco Oller (1833–1917), José Cuchí y Arnau (1857–1925), and Ramón Frade (1875–1954). The paintings, belonging to the Corporación de las Artes Musicales and Museo de Arte de Puerto Rico (San Juan, Puerto Rico), were investigated through a combination of complementary non- and micro-invasive scientific techniques. The use of non-invasive macro-X-ray fluorescence (MA-XRF) and reflectance imaging spectroscopy (RIS) was applied for the first time to characterize Puerto Rican artists’ palette. The non-invasive approach was integrated with spectroscopic techniques such as Raman and/or Fourier-transform infrared (FTIR) spectroscopy, as well as high pressure liquid chromatography coupled to diode array detector and tandem mass spectrometry (HPLC-DAD-MS/MS), when sampling was possible. While this technical investigation reveals pigments that are typical for late 19th/early twentieth century paintings, it also emphasizes some unexpected findings, including the use of cobalt green and synthetic yellow lakes, which enabled the date given to some of the paintings to be refined to post 1910 rather than their current dates of ca. 1890. This study confirms that the Puerto Rican artist’s palettes are very similar to their European contemporaries, underscoring both their European training and their attempt to adapt these methods of painting to a new Caribbean identity emerging from the Spanish American War.