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Background: A protocol exists for the collection of samples to investigate unexplained hypoglycaemia, termed the 'hypopack'. These packs are kept in Accident and Emergency departments and neonatal special care baby units throughout Northern Ireland and most wards of the Regional Children's Hospital. A retrospective audit of 107 hypopacks received between July 2001 and December 2003 highlighted a number of problems: samples collected when the patient was receiving dextrose, incomplete clinical history provided, insufficient and haemolysed samples received and poor filing of reports in charts. These were addressed by re-designing the request form, updating the protocol and introducing a summative report. The new protocol was introduced in April 2006. Methods: The aim of this study is to assess whether the revised protocol improved utility of the hypopack. A retrospective re-audit of 100 hypopacks received between April 2006 and May 2007 was performed. Results: Forty-nine percent of patients were hypoglycaemic (<2.6 mmol/L) compared with 35% in the original audit. In both audits, 33% of laboratory reports were missing from patients' charts. One case of medium-chain acyl-CoA dehydrogenase deficiency, three cases of hyperinsulinism and two endocrine-related cases were identified. Conclusions: The new hypopack protocol has increased the number of appropriately performed investigations. Provision of information concerning dextrose infusion has assisted the interpretation of the hypopack results. [ABSTRACT FROM AUTHOR]