Negative pressure wound therapy has been used to promote wound healing in a variety of settings, including as an adjunct to silver‐impregnated dressings in the acute management of paediatric burns. Fluid aspirated by the negative pressure wound therapy system represents a potentially insightful research matrix for understanding the burn wound microenvironment and the intervention's biochemical mechanisms of action. The aim of this study was to characterize the proteome of wound fluid collected using negative pressure wound therapy from children with small‐area thermal burns. Samples were obtained as part of a randomized controlled trial investigating the clinical efficacy of adjunctive negative pressure wound therapy. They were compared with blister fluid specimens from paediatric burn patients matched according to demographic and injury characteristics. Protein identification and quantification were performed via liquid chromatography tandem mass spectrometry and sequential window acquisition of all theoretical mass spectra data‐independent acquisition. Proteins and biological pathways that were unique to or enriched in negative pressure wound therapy fluid samples were evaluated using principal components, partial least squares‐discriminant, and gene ontology enrichment analyses. Eight viable samples of negative pressure wound therapy fluid were collected and analyzed with eight matched blister fluid samples. A total of 502 proteins were quantitatively profiled in the negative pressure wound therapy fluid, of which 444 (88.4%) were shared with blister fluid. Several proteins exhibited significant abundance differences between fluid types, with negative pressure wound therapy fluid showing a higher abundance of matrix metalloproteinase‐9, arginase‐1, low affinity immunoglobulin gamma Fc region receptor III‐A, filamin‐A, alpha‐2‐macroglobulin, and hemoglobin subunit alpha. The results lend support to the hypothesis that negative pressure wound therapy augments wound healing through the modulation of factors involved in the inflammatory response, granulation tissue synthesis, and extracellular matrix maintenance. Data are available via ProteomeXchange with identifier PXD023168.