Background: The use of cell-free hemoglobin (Hgb) solutions, such as Oxyglobin (Biopure Corp, Cambridge, MA, USA), as a blood substitute for the treatment of acute anemias is increasing in veterinary medicine. These solutions interfere with colorimetric tests, which do not discriminate between cellular Hgb (Hgbcell) from the patient and extracellular Hgb (Hgbdelta) from the Oxyglobin, and therefore make the monitoring of anemia, based on Hgb concentration, difficult. The ADVIA 120 hematology analyzer (Bayer Diagnostics, Tarrytown, NY, USA) evaluates Hgb by 2 methods, a standard cyanmethemoglobin colorimetric method and flow cytometry, and therefore might provide the means to differentiate extracellular and intracellular Hgb. Objective: The objective of this study was to determine the accuracy and precision of the ADVIA 120 in differentiating extracellular from intracellular Hgb. Methods: Anticoagulated whole blood samples from 10 healthy dogs were analyzed in triplicate on the ADVIA 120. Hgbdelta concentration was determined by adding Oxyglobin (13 g/dL) to the whole blood samples at dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. Hgbcell and Hgbtotal values were calculated and compared with actual values by linear regression. Analyses were done in triplicate and repeated 9 consecutive times to evaluate intra-assay precision of Hgbtotal and Hgbcell determinations. Results: Correlation between Hgb values obtained by colorimetric (Hbtotal) and flow cytometric (Hgbcell) methods on whole blood samples was high (R2= .99; n = 10) with a slope of 0.96 and intercept of 0. Correlation between actual and predicted Hgbcell values also was high (R2= .99), with a small positive bias (0.289 ± 0.185; n = 60). Intra-assay precisions were high, with most coefficients of variation <2%. Conclusion: The ADVIA 120 is capable of differentiating Hgbcell from Hgbdelta. The flow cytometric method is accurate and precise when compared with the cyanmethemoglobin method. A small bias between the results is unlikely to be clinically significant but may affect the ability of the ADVIA to differentiate small quantities (<0.3 g/dL) of Hgbdelta.