Background: Large-scale genomic studies of bladder cancer (BC) have identified MAPK as a recurrent pathway alteration. The MAPK pathway is a signaling cascade comprising of the proteins RAF, MEK, and ERK, which together control critical cellular processes. Currently, cisplatin-based chemotherapy and anti-PD1/PD-L1 agents are the backbone of systemic therapy in BC, yet only a subset of patients respond. As such, additional novel therapeutic strategies, such as targeting the MAPK pathway, are needed. Herein, we describe the impact of MAPK alterations on BC tumor properties sensitivity to novel MAPK-directed agents. Methods: The activity of the activity of RAF inhibitors (including RAF265 and the novel RAF dimerization inhibitor LXH254) was tested in BC cell lines with diverse MAPK pathway alterations including RAF1 amplification and NRAS mutations. Relative cell viability was measured by luminescence assay following treatment. RAF1-amplified UMUC9 cells were implanted as mouse flank xenografts which were randomized to treatment with vehicle or RAF inhibitor alone or in combination with a MEK inhibitor, trametinib. Mouse weights and tumor measurements were monitored during the treatment period and tumors were excised and weighed. C57BL/6 mice were orthotopically implanted with BBN cells with and without a RAF1 amplification using a surgical approach and tumor volumes were measured under ultrasound (US). Results: RAF1-amplified and NRAS-mutated cell lines demonstrated higher sensitivity to RAF inhibition than non-altered lines. There was a significant reduction in tumor volumes of UMUC9-engrafted mice treated with RAF265 (n=10) or RAF265 plus trametinib (n=8) compared to vehicle controls (n=14). Mice treated with RAF265 alone or with RAF265 plus trametinib had significantly lower tumor weights than vehicle-treated mice. US monitoring showed that mice orthotopically implanted with RAF1-amplified BBN cells (RAF1, n=9) and empty vector cells (EV, n=9) showed take rates [RAF1=67%, EV=78%], mean tumor sizes [RAF1=23.0mm², EV=12.5mm², p=0.07], and tumor growth rates [RAF1=0.60mm²/day, EV=0.40mm²/day, p=0.19]. Conclusions: We sought to explore the vulnerabilities incurred by MAPK pathway alterations in BC. We tested the activity of a RAF inhibitors in BC cell lines with RAF1 amplifications and NRAS mutations and found that MAPK pathway-altered models displayed increased sensitivity to RAF inhibition compared to MAPK-unaltered models. We further showed this sensitivity in vivo with RAF1-amplified mouse xenografts and elucidated the synergistic effect of concurrent RAF and MEK inhibition. There was a trend towards larger tumors and more rapid growth rate in orthotopically-implanted RAF1-amplified BC cells compared to controls. Taken together, these data identify MAPK pathway alterations as a novel dependency in BC that may be clinically targeted with small molecule inhibitors.