Abstract Objectives The estimates of biological variation (BV) have traditionally been determined using direct methods, which present limitations. In response to this issue, two papers have been published addressing these limitations by employing indirect methods. Here, we present a new procedure, based on indirect methods that analyses data collected within a multicenter pilot study. Using this method, we obtain CV I estimates and calculate confidence intervals (CI), using the EFLM-BVD CV I estimates as gold standard for comparison. Methods Data were collected over a 18-month period for 7 measurands, from 3 Spanish hospitals; inclusion criteria: patients 18–75 years with more than two determinations. For each measurand, four different strategies were carried out based on the coefficient of variation ratio (rCoeV) and based on the use of the bootstrap method (OS1, RS2 and RS3). RS2 and RS3 use symmetry reference change value (RCV) to clean database. Results RS2 and RS3 had the best correlation for the CV I estimates with respect to EFLM-BVD. RS2 used the symmetric RCV value without eliminating outliers, while RS3 combined RCV and outliers. When using the rCoeV and OS1 strategies, an overestimation of the CV I value was obtained. Conclusions Our study presents a new strategy for obtaining robust CV I estimates using an indirect method together with the value of symmetric RCV to select the target population. The CV I estimates obtained show a good correlation with those published in the EFLM-BVD database. Furthermore, our strategy can resolve some of the limitations encountered when using direct methods such as calculating confidence intervals.