This study was designed to compare the performance of the kit Hemacolor (HC) in two protocols (A, B) and Toluidine blue stain (TB) for staining the bull sperm head in samples of frozen-thawed semen. Automated Sperm Morphology Analysis (ASMA) was performed to determine the sperm measurements: head size (length, width, area and perimeter). TB was found to be the best procedure for staining the frozen-thawed bull sperm (p<0.0001). The use of this method rendered the highest number of cells correctly analyzed (88.29%) and the lowest coefficient of variation on the image processing (4.54) and morphometric measurements. TB provided good colour intensity and optimum contrast of the sperm head with the surrounding background that allows efficient boundary detection and reduces the number of stained foreign particles. The staining methods affected significantly the sperm head dimensions (p<0.0001) with increased values from HC (protocol A) than HC (protocol B) and TB, respectively (HC>TB). HC provide more intense grey-level values, resulting in enlarged cells, which influence the size morphometric parameters. Based on these findings, we recommend TB for its accurate and reproducible morphometric results.