Stability of concentrations of urinary stone-related metabolites was analyzed from samples of recurrent urinary stone formers to assess necessity and effectiveness of urine acidification during collection and storage. First-morning urine was collected from 20 adult calcium-stone forming patients at Tomas Bata Hospital in the Czech Republic. Urine samples were analyzed for calcium, magnesium, inorganic phosphate, uric acid, sodium, potassium, chloride, citrate, oxalate, and urine particles. The single-voided specimens were collected without acidification, after which they were divided into three groups for storage: samples without acidification ("NON"), acidification before storage ("PRE"), or acidification after storage ("POST"). The analyses were conducted on the day of arrival (day 0, "baseline"), or after storage for 2 or 7days at room temperature. The maximum permissible difference (MPD) was defined as ±20 % from the baseline. The urine concentrations of all stone-related metabolites remained within the 20 % MPD limits in NON and POST samples after 2days, except for calcium in NON sample of one patient, and oxalate of three patients and citrate of one patient in POST samples. In PRE samples, stability failed in urine samples for oxalate of three patients, and for uric acid of four patients after 2days. Failures in stability often correlated with high baseline concentrations of those metabolites in urine. Detailed procedures are needed to collect urine specimens for analysis of urinary stone-related metabolites, considering both patient safety and stability of those metabolites. We recommend specific preservation steps.