Background: Current guidelines recommend monitoring the adequacy of hemodialysis (HD) treatments in patients with acute kidney injury (AKI). Blood-based methods for calculating urea such as reduction ratio (URR) and single-pool Kt/Vurea (spKt/Vurea) require pre- and post-HD blood urea nitrogen (BUN) measurements. This study aims to compare real-time monitoring of urea clearance using dialysate ultraviolet absorbance (UV) with laboratory-measured spKt/Vurea. Methods: We conducted a single-center, retrospective study among hospitalized patients with AKI, who required intermittent hemodialysis (IHD). Those patients whose dialysis dose was simultaneously monitored by spKt/Vurea and UV-absorbance (UV-spKt/Vurea) were included in the study. The statistical correlation between both methods was assessed by means of the Pearson moment product correlation, Mann–Whitney U-test and Bland–Altman analysis of agreement. Results: Thirty patients with AKI were evaluated. There was no statistical difference between the mean spKt/Vurea calculated by traditional methods and the mean UV-spKt/Vurea. (1.37 ± 0.37 vs. 1.28 ± 0.36, P = 0.12, CI: 95%). A Pearson moment correlation analysis revealed a close agreement between both methods (r = 0.79, P < 0.001). Furthermore, Bland–Altman analysis showed that >95% of the data points were confined within the upper and lower levels of agreement. Conclusion: In this pilot study of patients with AKI, UV-spKt/Vurea correlated with standard blood-based spKt/Vurea and may be a useful tool to monitor dialysis adequacy. Larger studies evaluating multiple UV and blood-based measurements per patient and a more diverse AKI population are needed to confirm this initial observation.