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Background The adenosine deaminase (ADA) enzyme is a marker of inflammatory processes whose activity can be measured through a colorimetric method developed as an in‐house assay. This validation can reduce costs and expand the alternatives for laboratory diagnosis. Methods The ADA analysis was achieved through a modified form of Giusti and Galanti's (1984) method. The following parameters were characterized: calibration curve, linearity, analytical sensitivity, limit of detection, limit of quantification, method working range, precision (within‐assay and between‐assay), bias, total analytical error, and sample stability. The results were statistically evaluated and compared with quality specifications based on biological variations and the performance of commercial tests. Results The analytical sensitivity and limit of detection (0.013 and 3.0 U/L, respectively) were lower than those of commercial tests. The method's working range was 3.2‐100.0 U/L. According to the quality specification, the method showed optimum performance with a bias <3.5%. However, repeatability (2.2% and 1.7% for normal‐ and high‐activity samples, respectively) and reproducibility achieved worse results when compared to commercial tests. The method demonstrated an inappropriate between‐assay precision for low enzymatic activity (10.4%) and the minimum and desirable performance for medium (8.8%) and high (5.0%) activities, respectively. It also presented at least a minimum performance (<25%) for the total analytical error of the three analyzed samples. The pleural fluid samples were found to be stable at −20°C for six days. Conclusion The findings show that the in‐house method displays an acceptable performance and is capable of generating results comparable to existing commercial tests.