Listeria monocytogenes is the causative agent responsible for listeriosis, one of the most severe foodborne diseases. Zero tolerance regulatory policies for the presence of L. monocytogenes in certain foods and the expensive recalls associated with contaminated foods require the need for rapid and cost-effective methods of detection of Listeria from food. The current study explored the ability of a matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) based proteomic approach to detect L. monocytogenes directly from selective enrichment broth containing spiked food, obviating the need for culturing on solid media. Pilot experiments were carried out with enriched spiked UHT milk (model food) in a non-selective enrichment broth. The limit of detection post-enrichment for obtaining species level identification using MALDI-TOF MS was determined as approximately 107-108 cfu/mL of enrichment broth. After obtaining successful species level identification with a starting inoculum of 1 cfu/mL, a 30 h detection scheme was developed for detection from an enrichment broth selective for Listeria. UHT milk spiked with single and mixed species cultures were incubated in the selective enrichment broth for 24 h, followed by another 6 h selective secondary enrichment. Further, the same 30 h scheme was applied to solid foods previously implicated in listeriosis, namely chicken pâté, cantaloupe and Camembert cheese. The scheme successfully detected as few as 1 and 10 cfu of bacteria per mL of initial selective broth in UHT milk and the solid foods, respectively. Thus, the proposed scheme of detection is simple, rapid and cost-effective compared to conventional methods used in diagnostic food microbiology laboratories. This study provides proof-of-concept evidence that MALDI-TOF MS can be used to detect L. monocytogenes directly from selective enrichment broth.