In dogs, diagnosis of incomplete ejaculation and azoospermia can be made by measuring the activity of the enzyme alkaline phosphatase (AP) in seminal plasma. However, even though upper cut-off value of 5000 IU / l is given in the literature, results by different assays may vary considerably. Furthermore, no data exist concerning the stability of the enzyme during storage of frozen seminal plasma, and no recommendations for pre-analytic dilutions can be found. During the present study, we compared results from a conventional large scale wet chemistry analyzer to a widely used dry chemistry point of care system (POC) and established a best practice for pre-analytical dilutions. Furthermore, stability of enzyme activities in seminal plasma during storage at −18°C for 24 h was evaluated. The average activity of AP in the 2nd fraction of normal ejaculates measured by Reflotron® was 107 328 IU / l. After 24 h of frozen storage, activities did not differ significantly (96 844 IU / l, p > 0.05). Fresh and frozen samples were analysed in parallel by the POC and conventional chemistry analyser, and the results compared that did not reveal a significant difference (p > 0.05). A dilution of seminal plasma with physiologic saline 1 : 100 prior to analysis was sufficient for the qualitative information whether AP activity is below or above 5000 IU / l. Present data show that AP measurement by a POC dry chemistry system is sufficiently accurate in diluted seminal plasma for the diagnosis of azoospermia and that seminal plasma can be stored frozen for 24 h before analysis.