Anterior cruciate ligament (ACL) and associated knee injuries severely impact on an individual’s opportunity to resume previous physical activity levels and may also increase the risk of developing knee osteoarthritis. While magnetic resonance imaging provides excellent diagnostic information on the specific structures involved in the injury, it is expensive and therefore not practical for providing repeated follow-up information after ACL reconstruction. Recent advances in protein profiling approaches including liquid chromatography–tandem mass spectrometry (LC–MS/MS) provides evidence to suggest that novel diagnostic biomarkers of knee injury and recovery may be detectable. Hence, this research focuses on interrogating the urinary proteome, using a quantitative data-independent acquisition mass spectrometry based proteomics technique (SWATHTM), to reliably quantify urinary proteins associated with acute ACL injury and repair status post-surgery. The MS data obtained from pre- and post-operative urine samples from 14 confirmed ACL injured patients were analysed and resulted in spectral library generation of more than 500 proteins. Multivariate statistical analysis revealed proteins that were associated with the injured cohort and proteins that could be indicative of tissue recovery outcomes. In addition, the potential influence of the pre-operative injury period on the protein abundance was investigated. The results indicated that the pre-operative injury period may affect the levels of a subset of proteins. Significantly overrepresented biological process, cellular component and molecular function gene ontologies related to these candidate proteins were also determined. Such biomarkers may provide additional tools for clinicians in the evaluation of both the initial injury but importantly, tissue recovery following surgery.