The Female Sexual Function Index is one of the most common instruments used to evaluate the female sexual function. The present study aimed to analyze the test-retest reliability, internal consistency, and discriminant validity of the index in clinical samples and to determine a specific and sensitive cutoff point for the Spanish version of the Female Sexual Function Index. For that purpose, a sample consisting of 117 Colombian women was recruited to evaluate test-retest reliability, and a second sample, consisting of 185 women, was divided into diagnosis and no-diagnosis groups based on DSM-5 criteria. Results showed adequate test-retest reliability after four weeks, and satisfactory evidence of internal consistency was obtained for subscale and overall scores. The inventory was found to have an adequate criterion validity, and it confirmed the differences between diagnosis and no-diagnosis groups. The instrument's cutoff point was determined to be 26 points, with a specificity of 73.9%, a sensitivity of 87.7%, and an area under the curve of 85.9 (CI = 80.0–91.7). These results confirm that the Spanish version of the FSFI is an adequate tool for evaluating female sexual dysfunction based on DSM-5 criteria.