Trypanosoma cruzi serological screening is recommended for people potentially exposed to this parasite in countries where Trypanosoma cruzi is endemic and those where it is not endemic. Blood samples on filter paper may be a practical alternative to plasma/serum for antibody detection. Using the Architect Chagas assay, we detected the presence of IgG against T. cruzi in matched serum and dried blood spots (DBS) collected from 147 patients residing in Madrid, Spain, who had potential previous exposure to T. cruzi. The κ statistic for the DBS/serum proportion of agreement for the detection of antibodies against T. cruzi was 0.803, considering an S/CO (assay result unit; chemiluminescent signal from the sample [S] divided by the mean chemiluminescent signal for the three calibrators used in the test [CO]) cutoff value of ≥1.00. The relative sensitivity of the Architect test using DBS increased from 95.2% to 98.8% when the cutoff was lowered from ≥1.00 to ≥0.88, while the relative specificity decreased from 84.1% to 71.6%. Overall, the median S/CO values for DBS were significantly lower than those for serum (2.6 versus 6.5; P < 0.001). Discrepancies that occurred with the use of DBS included 10 false positives (with low S/CO values in 9 cases [median, 2.13]) and 4 false negatives, with mean S/CO values of 0.905 (gray zone). Using DBS plus a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) may be a simple and reliable method for detecting IgG against T. cruzi when blood sampling by venipuncture is not feasible. This method may also reduce the false-negative rates observed with some rapid diagnostic tests. The lower relative sensitivity compared to the reference method may be increased by lowering the optical density threshold.