•Two-concentration linear regression is reliable for regulated LC–MS bioanalysis.•Two-concentration linear regression can typically save 15–20% of time and cost.•A minimum of six different concentrations is not necessary for linear regression. Linear calibration is usually performed using eight to ten calibration concentration levels in regulated LC–MS bioanalysis because a minimum of six are specified in regulatory guidelines. However, we have previously reported that two-concentration linear calibration is as reliable as or even better than using multiple concentrations. The purpose of this research is to compare two-concentration with multiple-concentration linear calibration through retrospective data analysis of multiple bioanalytical projects that were conducted in an independent regulated bioanalytical laboratory. A total of 12 bioanalytical projects were randomly selected: two validations and two studies for each of the three most commonly used types of sample extraction methods (protein precipitation, liquid–liquid extraction, solid-phase extraction). When the existing data were retrospectively linearly regressed using only the lowest and the highest concentration levels, no extra batch failure/QC rejection was observed and the differences in accuracy and precision between the original multi-concentration regression and the new two-concentration linear regression are negligible. Specifically, the differences in overall mean apparent bias (square root of mean individual bias squares) are within the ranges of −0.3% to 0.7% and 0.1–0.7% for the validations and studies, respectively. The differences in mean QC concentrations are within the ranges of −0.6% to 1.8% and −0.8% to 2.5% for the validations and studies, respectively. The differences in %CV are within the ranges of −0.7% to 0.9% and −0.3% to 0.6% for the validations and studies, respectively. The average differences in study sample concentrations are within the range of −0.8% to 2.3%. With two-concentration linear regression, an average of 13% of time and cost could have been saved for each batch together with 53% of saving in the lead-in for each project (the preparation of working standard solutions, spiking, and aliquoting). Furthermore, examples are given as how to evaluate the linearity over the entire concentration range when only two concentration levels are used for linear regression. To conclude, two-concentration linear regression is accurate and robust enough for routine use in regulated LC–MS bioanalysis and it significantly saves time and cost as well.