Patients with childhood hypophosphatasia (HPP) often have unspecific symptoms. It was our aim to identify patients with mild forms of HPP by laboratory data screening for decreased alkaline phosphatase (AP) within a pediatric population. We conducted a retrospective hospital-based data screening for AP activity below the following limits: Girls:≤12 years: <125U/L; >12 years: <50U/L Boys:≤14 years: <125U/L; >14 years: <70U/L. Screening positive patients with otherwise unexplained hypophosphatasemia were invited for further diagnostics: Re-test of AP activity, pyridoxal 5'-phosphate (PLP) in hemolyzed whole blood, phosphoethanolamine (PEA) in serum and urine, and inorganic pyrophosphate in urine. Sequencing of the ALPL gene was performed in patients with clinical and/or laboratory abnormalities suspicious for HPP. We assessed a total of 14,913 samples of 6,731 patients and identified 393 screening-positive patients. The majority of patients were excluded due to known underlying diseases causing AP depression. Of the 30 patients who participated in the study, three had a decrease in AP activity in combination with an increase in PLP and PEA. A heterozygous ALPL mutation was detected in each of them: One patient with a short stature was diagnosed with childhood-HPP and started with enzyme replacement therapy. The remaining two are considered as mutation carriers without osseous manifestation of the disease. A diagnostic algorithm based on decreased AP is able to identify patients with ALPL mutation after exclusion of the differential diagnoses of hypophosphatasemia and with additional evidence of increased AP substrates.