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Direct analysis of whole blood on bloodstained textiles is achieved with thread spray mass spectrometry (MS). This capability satisfies investigators' first priority in crime scene investigations, which is determining if a stain is blood. This thread spray method explores the use of evidentiary fabric threads for rapid determination of hemoglobin directly from whole blood within textiles without prior extraction steps. The multiplicity of information that can be derived from the thread spray MS method distinguishes it from the current presumptive Bluestar® method, by enabling the detection of hemoglobin (both α- and β-chains), the heme co-factor and lipids all from a single blood sample. Lipid composition was found to differ for blood samples originating from human, canine, and horse species. The robustness of the thread spray MS method as a forensic analytical platform was evaluated in three ways: (1) its successful applicability to samples previously tested by the Bluestar® presumptive method, offering a confirmatory test without prior sample pre-treatment, (2) successful detection of heme from previously washed fabrics, which demonstrated the unprecedented sensitivity of the thread spray method, and (3) the ability to analyze samples stored under ambient conditions for up to 30 days. These results attest to the potential capabilities of the thread spray MS platform in forensic serology, and its application for direct analysis of evidentiary garments, which confer the advantages of rapid analysis and the reduction of the false positive and negative identification rates for blood on textiles.