Introduction: Diagnostic practice for acute abdominal pain at the Emergency Department varies widely and is mostly based on doctor's preferences. We aimed at developing an evidence-based guideline for the diagnostic pathway of patients with abdominal pain of non-traumatic origin. Methods: All available international literature on patients with acute abdominal pain was identified and graded according to their methodological quality by members of the multidisciplinary steering group. A guideline was synthetized, providing evidence-based recommendations together with considerations based on expertise of group members, patient preferences, costs, availability of facilities, and organizational aspects. Conclusions and Recommendations: Definition: Uniform terminology is needed in patients with acute abdominal pain to avoid difficulty in interpretation and ease comparison of findings between studies. We propose the use of the following definition for acute abdominal pain: pain of nontraumatic origin with a maximum duration of 5 days. Clinical diagnosis: Clinical evaluation is advised to differentiate between urgent and nonurgent causes. The diagnostic accuracy of clinical assessment is insufficient to identify the correct diagnosis but can discriminate between urgent and nonurgent causes. Patients suspected of nonurgent diagnoses can safely be reevaluated the next day. Based on current literature, no conclusions can be drawn on the differences in accuracy between residents and specialists. No conclusions can be drawn on the influence of a gynecological consultation. In patients suspected of an urgent condition, additional imaging is justified. CRP and WBC count alone are insufficient to discriminate urgent from nonurgent diagnoses. Diagnostic imaging: There is no place for conventional radiography in the work-up of patients with acute abdominal pain due to the lack of added value on top of clinical assessment. Computed tomography leads to the highest sensitivity and specificity in patients with acute abdominal pain. Positive predictive value of ultrasound is comparable with CT and therefore preferred as the first imaging modality due to the downsides of computed tomography; negative or inconclusive ultrasound is followed by CT. Based on current literature, no conclusions can be drawn on the added value of a diagnostic laparoscopy in the work-up of patients with acute abdominal pain. Antibiotic treatment should be started within the first hour after recognition of sepsis. Administration of opioids (analgesics) decreases the intensity of the pain and does not affect the accuracy of physical examination.