Targeted proteomics has become the method of choice for biomarker validation in human biopsies due to its high sensitivity, reproducibility, accuracy, and precision. However, for targeted proteomics to be transferred to clinical routine there is the need to reduce its complexity, make its procedures simpler, increase its throughput, and improve its analytical performance. Here we present the Isotopologue Multipoint Calibration (ImCal) quantification strategy, which uses a mix of isotopologue peptides to generate internal multipoint calibration curves for each individual sample and to accurately quantify biomarker peptides in clinical applications without the need of expert supervision. ImCal relies on the use of five different isotopically-labelled peptides of different nominal mass mixed at different concentrations to be used as an internal calibration curve for each endogenous peptide. The use of internal multipoint calibration curves is well-suited for the generation of ready-to-use biomarker kits for clinical applications as it is compatible with both high- and low-resolution mass spectrometers and different levels of endogenous peptide, it eliminates the need for blank matrixes required in external curves, it allows the evaluation of matrix effects and the valid quantification range in each individual sample, and it does not require expert adjustment. We used the ImCal method to quantify HER2 in 35 breast cancer formalin-fixed paraffin-embedded patient samples, revealing a high degree of heterogeneity among patients, which contrasts with the homogeneous immunohistochemistry patient classification. Our work illustrates how an improvement of mass spectrometry methods for biomarker quantification can provide fine-grain patient stratification, and thus better disease diagnostic and prognosis.