The activity of proteins rather than the concentration of proteins in biopharmaceutical and in vitro diagnostics are often the primary focus. Nonetheless, development of a calibration-free concentration analysis (CFCA) approach that accurately quantifies the concentration of proteins based on molecular interactions with specific monoclonal antibodies and without the requirement of external calibrators would be beneficial to diagnostics. Generally, only analytes that interact with the antibody (Ab) are quantified by CFCA. Moreover, protein concentrations measured by CFCA usually vary when different Abs are used, and are lower than those obtained by amino acid analysis because any non-native state population of the target protein is not captured by the Ab. To achieve comparable results between CFCA and traditional amino acid analysis (AAA), an Ab that recognizes the target protein irrespective of its conformation should be used. In this report, three different monoclonal antibodies were used to quantify purified human myoglobin in solution by CFCA. The concentrations obtain by the Abs (i.e., 2.985, 2.912, 3.032 mg mL−1) were comparable with that obtained by AAA. Moreover, isotope dilution mass spectrometry (IDMS) gave a human myoglobin concentration of 2.851 mg mL−1, which is also in agreement with the results from CFCA. The performance of CFCA was evaluated by measuring various parameters, including within-day and between-day precision. The results demonstrated that the active concentration measured by CFCA is comparable with that of IDMS when the appropriate Ab is used. Recommended procedures for performing the new CFCA approach are provided. This study shows that CFCA represents a primary method for accurate protein concentration determination, which should aid the development of certified reference materials.