Background Alkaline phosphatase isoenzyme analysis is an expensive and time-consuming laboratory test. We evaluated the effect of a locally derived screening algorithm for alkaline phosphatase isoenzyme requests on the number of alkaline phosphatase isoenzyme analyses performed, laboratory cost and patient care. Method A total of 110 alkaline phosphatase isoenzyme analysis requests from the year 2015 were reviewed and subsequent alkaline phosphatase concentrations were monitored over a two-year period, to determine if the decision of performing/not performing alkaline phosphatase isoenzyme analysis, based on the algorithm, had an impact on patient care and laboratory cost. All alkaline phosphatase isoenzyme analysis requests with two consecutive elevated alkaline phosphatase concentrations (>upper limit of reference interval) were screened and, subject to the gamma glutamyl transferase being within the reference interval, either Bone alkaline phosphatase or 25 hydroxyvitamin D was measured depending on the age of the patient. Results Compliance with this algorithm led to the normalization of subsequent serum alkaline phosphatase in 97% of patients without requiring alkaline phosphatase isoenzyme analysis. The cost of performing Bone alkaline phosphatase and 25 hydroxyvitamin D in-house was £778.50, while the cost of performing alkaline phosphatase isoenzyme analysis would have been £3040. This resulted in a laboratory saving of £2261.50. Conclusions Implementation of this algorithm led to a significant reduction in alkaline phosphatase isoenzyme analysis, without compromising patient care. Total savings could be increased if 25 hydroxyvitamin D was used as a first-line test, for all patients with an elevated alkaline phosphatase and a normal gamma glutamyl transferase regardless of age. This algorithm is cost-effective and can be implemented in laboratories with 25 hydroxyvitamin D assay.