BACKGROUND In veterinary clinical laboratories, qualitative tests for total protein measurement in canine cerebrospinal fluid (CSF) have been replaced by quantitative methods, which can be divided into dye-binding assays and turbidimetric methods. There is a lack of validation data and reference intervals (RIs) for these assays. OBJECTIVES The aim of the present study was to assess agreement between the turbidimetric benzethonium chloride method and 2 dye-binding methods (Pyrogallol Red-Molybdate method [PRM], Coomassie Brilliant Blue [CBB] technique) for measurement of total protein concentration in canine CSF. Furthermore, RIs were determined for all 3 methods using an indirect a posteriori method. METHODS For assay comparison, a total of 118 canine CSF specimens were analyzed. For RIs calculation, clinical records of 401 canine patients with normal CSF analysis were studied and classified according to their final diagnosis in pathologic and nonpathologic values. RESULTS The turbidimetric assay showed excellent agreement with the PRM assay (mean bias 0.003 g/L [-0.26-0.27]). The CBB method generally showed higher total protein values than the turbidimetric assay and the PRM assay (mean bias -0.14 g/L for turbidimetric and PRM assay). From 90 of 401 canine patients, nonparametric reference intervals (2.5%, 97.5% quantile) were calculated (turbidimetric assay and PRM method: 0.08-0.35 g/L (90% CI: 0.07-0.08/0.33-0.39); CBB method: 0.17-0.55 g/L (90% CI: 0.16-0.18/0.52-0.61). Total protein concentration in canine CSF specimens remained stable for up to 6 months of storage at -80°C. CONCLUSIONS Due to variations among methods, RIs for total protein concentration in canine CSF have to be calculated for each method. The a posteriori method of RIs calculation described here should encourage other veterinary laboratories to establish RIs that are laboratory-specific.