Abstract Background The measurements of Calcium (Ca) and Phosphorus (P) in 24-hour urine samples are associated with the evaluation of hyperparathyroidism, renal tubular acidosis, and monitoring of Falconi Syndrome and Nephrolithiasis. The aim is to demonstrate the maintenance of analytical performance without the addition of 6N hydrochloric acid (HCl) in the pre-analytical process. Methods The study was divided into stages and carried out for both analytes. In the first stage, correlation tests were performed on 40 samples with and without HCl within the range of linearity. Imprecision was processed at 3 clinically relevant concentrations for 5 days, repeated 5 times. In the second stage, stability testing of Ca and P was performed on 7 samples over 5 days, without HCl (pH 5–6.7), with HCl (pH < 3), alkalized (pH > 8), and neutral (pH 7–7.5). In the last stage, interferent testing was performed on 286 samples, where pH was measured after collection and after acidification with 200uL of HCl. A pH meter and pH strips were used to measure pH, and a Siemens® - Advia 2400 equipment was used for analytical measurement of Ca and P. Results In the correlation results, the variance test for both analytes resulted in F-test: 1.01 < 1.69, the difference analysis was less than 2.02, correlation coefficient and Bias% of 1.00 and 0.55 for Ca and 0.99 and 0.27 for P, respectively. Total error (TE) obtained for Ca was 9.69 and 9.41, lower than the allowed TE for both analytes. The repeatability and intralaboratory imprecision of the three levels of each analyte were less than 13.8% for Ca and 9.0% for P. There was 100% correlation of the results when the analyses were performed with samples at pH up to 7; at pH values > 7, the concentrations decreased by 60%. All 268 samples with different pH values, subjected to 200uL of HCl, were suitable for Ca and P measurement with pH below 5.7. Conclusions Samples with a pH greater >or = to 7 necessarily need to be acidified with HCl 6N. There is no analytical alteration if all samples in the routine of urinary Ca and P are submitted to 200uL of HCl 6N, even the most alkaline samples. The non-provision of preservative containers to patients reduces the risk of accidents, costs, and the need for legal licenses for handling in healthcare facilities.