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This study evaluates quantitative combined single photon emission tomography/computed x-ray tomography (SPECT/CT) to assess the influence of radiotracer concentration on detection of sentinel lymph nodes (SLN) in endometrial cancer (EC). Eighty EC patients underwent pericervical 99m-Tc-nanocolloid injection followed by SPECT/CT. The subgroup of patients with failed SLN detection in SPECT/CT (<i>n</i> = 20) was compared to match-paired patients showing at least two SLNs. Results of intraoperative gamma probe measurements and quantitative SPECT/CT were used for comparison. In patients with detection failure, the percentage of injected dose, absolute activity, and volume of the injection site were significantly lower (30 ± 24% vs. 55 ± 19%; <i>p</i> = 0.01, 43 ± 36 MBq vs. 73 ± 33 MBq; <i>p</i> = 0.04, and 183 ± 164 mL vs. 266 ± 164 mL; <i>p</i> = 0.03) while mean activity concentration in liver, spleen, and bone marrow was significantly higher (<i>p</i> = 0.02). Activity concentration (>33 KBq/mL) and absolute activity (>73 MBq) of injection areas correlated with successful intraoperative SLN detection. In a subgroup of 19 SLNs, a correlation between SPECT/CT-derived uptake and intraoperative count rate was found (R² = 0.8; <i>p</i> < 0.001). SLN detection in EC patients depended on high radiotracer activity at the cervical injection site. Quantitative SPECT/CT could predict successful intraoperative SLN detection and may help to optimize injection technique.