GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl) guanine (PMEG) has significant activity as monotherapy in dogs with non-Hodgkin's lymphoma. Phase I trials have been initiated in humans based on the encouraging activity observed in canine lymphoma. Two new analogues of GS-9219 (GS-343074 and GS-424044) were recently produced for evaluation as potential novel antineoplastic agents against solid tumours. As a preclinical step, effect of GS-343074 and GS-424044 were evaluated against ten canine cancer cell lines for antiproliferative effect. Both analogues displayed antiproliferative activity against multiple canine cancer cell lines, although GS-343074 was more potent and of broader spectrum compared to GS-424044. Flow cytometric analysis of cells that experienced growth inhibition support apoptotic death as a mechanism of action for both analogues. On the basis of in vitro results described here, GS-343074 and GS-424044 show promise as novel anticancer agents in canine cancer.