In 2018 a novel influenza polymerase inhibitor antiviral, Baloxavir Marboxil (BXM), was licensed in Japan and the US. Baloxavir acid (BXA), the active metabolic form of BXM, binds to the endonuclease region of the PA subunit in influenza A and B viruses. The amino acid substitutions I38T, I38M and I38F in the PA gene have been reported to confer reduced susceptibility to BXM and shown to arise at a frequency of 2.2-23% in patients receiving BXM treatment. Two important factors are currently unknown, the fitness of the I38T/M/F variants and the potential for other PA amino acid substitutions to arise that have reduced BXM susceptibility. To better understand these questions, BXM susceptibility monitoring of circulating influenza viruses should be conducted. We have developed a cell culture-based focus reduction assay to determine the susceptibility of 286 seasonal influenza viruses circulating prior to the licensure of BXM. This includes influenza viruses of A(H1N1)pdm09 and A(H3N2) subtypes, B/Yamagata and B/Victoria lineages, and viruses with neuraminidase inhibitor resistance. BXA was effective against all influenza subtypes tested with mean EC50 values (minimum-maximum) of 0.7 ± 0.5 nM (0.1-2.1 nM), 1.2 ± 0.6 nM (0.1- 2.4 nM), 6.7 ± 4.6 nM (0.7-14.8 nM), and 7.2 ± 3.5 nM (1.8-15.5 nM) obtained for A(H1N1)pdm09, A(H3N2), B/Victoria lineage, and B/Yamagata lineage influenza viruses, respectively. We utilised reverse genetics to introduce I38T, I38M, I38F PA substitutions into A(H1N1)pdm09, A(H3N2) and B viruses. Our assay was capable of measuring reduced BXA susceptibility in the reverse genetics derived variant viruses, with increased EC50 values of 5 to 65-fold depending on the virus and substitution compared to wild type. With BXM now licensed in two countries this assay is likely to be utilised in the coming 2019 influenza season in the Southern Hemisphere to monitor for viruses with reduced BXM susceptibility.