Coffee consumption habits have been reported to directly impact the liking of bitterness. Bitter compounds in coffee have also been linked to gastroesophageal reflux disease. Consequently, understanding the molecular basis of coffee bitterness is of interest. Bitter compounds in coffee have been identified using taste-guided analytical approach; however, the sensory relevance of these compounds and their contribution to overall bitterness in coffee are not well established. The overall objective of the current project was to apply an untargeted chemical profiling approach (flavoromics) to identify chemical drivers of coffee bitterness. A total of 14 commercial roasted coffee beans were selected from the market. Their respective coffee brews were chemically profiled using UPLC/MS-QTOF. Sensory analysis by 8 trained panelists was carried out to evaluate the bitter intensity of same brews. The chemical profiles and the corresponding bitter intensity ratings were modeled by orthogonal partial least squares (OPLS) with good fit (R2Y > 0.9) and predictive ability (Q2 > 0.9). Thirteen chemical markers that highly correlated to bitter intensity were subsequently isolated by multi-dimensional preparative LC/MS and further identified by MS and NMR. The sensory recombination test of negative markers demonstrated significant reduction on coffee bitterness. Sensory investigation and structural elucidation of individual negative markers are currently ongoing. This work will provide new understanding of the chemistry driving overall coffee bitterness and allow the industry to tailor coffee products to different consumer preferences.