Introduction: Anastomotic leak (AL) is the anathema of colorectal surgery. Early diagnosis is important to enable early intervention. Temporary diverting ileostomy (TDI) does not prevent AL and presents inherent complications. Numerous drain fluid biomarkers have been studied in colorectal surgery and extravasated intraluminal substances such as lipase have shown promise. The aim of this study was to assess drain fluid lipase (DFL) as a biomarker of AL after rectal resection with low colorectal anastomosis and no TDI. Methods: This prospective observational cohort study collected samples of DFL in consecutive patients undergoing rectal resection with low colorectal anastomosis and without TDI. Data were collected and analysed relating to patient demographics, operation, indication and daily DFL measurements from pelvic drains. Results: Between August 2018 and March 2021, there were 62 patients recruited who met eligibility criteria. Overall, the anastomotic leak rate was 9.7% (6/62). These six patients were assigned to the AL group. The DFL on postoperative day 3 was found to be higher in the AL group compared to the NAL group (p = 0.034). A DFL cutoff threshold of less than 64 U/L was found to have a negative predictive value of 97.6% and negative likelihood ratio of 0.32 in excluding AL. Conclusion: This is the first study is to assess the utility of DFL as a biomarker for AL following colorectal anastomosis without TDI. The measurement of DFL has value as a strong negative predictor of AL and moderate impact on post-test probability. The results are comparable to the use of systemic biomarkers.