Aims: This study aimed to analyze and compare the quantity of energy and fat using the infrared analysis and creamatocrit method in pasteurized human milk (HM) samples. Methods: This cross-sectional study analyzed 1,858 pasteurized human samples from 317 mothers at a single center. Infrared transmission spectrophotometry (Miris, Human Milk Analyser [HMA], Uppsala, Sweden) and the creamatocrit method were used to evaluate the quantity of energy and fat in pasteurized HM samples. Results: The average age of donor mothers was 29.7 ± 5.1 years, and the median duration of lactation was 22 days (interquartile range [IQ]: 7.7-59.2). Full-term births were observed in 196 (95.1%) of the women. The values of energy (difference: +8.96 kcal/dL, 95% CI: 8.52-9.44 kcal/dL; p < 0.001) and fat (difference: +0.40 g/dL, 95% CI: 0.35-0.45 g/dL; p < 0.001) in HM samples obtained by Miris were higher than those by the creamatocrit method. The energy calculated and the fat measured by Miris in the HM samples correlated moderately and directly with the obtained by creamatocrit (fat, r = 0.585; p < 0.001 and energy, r = 0.591; p < 0.01). The linear regression, adjusted for maternal age and lactation time, showed that the energy values calculated by creamatocrit were directly associated with those of Miris (energy kcal/dL = 38.43 + [0.516 × kcal/dL of creamatocrit]). Conclusion: The energy and fat quantity of pasteurized HM samples obtained by the creamatocrit and infrared methods were significantly correlated. However, the values calculated by the creamatocrit method were significantly lower than those by the infrared analyzer.