Acute diarrheal disease (ADD) is the most frequent cause of childhood illness and a leading cause of death in children less than 5 years of age [1]. In 2010, there were 1.731 billion episodes of diarrhea that lead to 700,000 deaths globally, mainly in low and middle income countries [2]. Most episodes of ADD are relatively mild to moderate; however about 2% of cases lead to severe diarrhea that is a risk factor for significant morbidity and mortality [2]. In addition to the risk of mortality from severe cases, recurrent episodes of ADD increase the risk of malnutrition. A recent study showed that odds of stunting increase by 1.13 times for every 5 episodes of diarrhea in children less 2 years of age [3]. Stunting is an important nutritional indicator as it represents long-term nutritional deficiency and is associated with cognitive deficits [2, 4]. In rare cases, selective enteropathogens like E. coli and Campylobacter can lead to severe morbidities (irrespective of severity of diarrhea), like hemolytic uremic syndrome and Guillain-Barre syndrome, among other conditions [5, 6]. The etiology and incidence of ADD vary in different regions of the world. The “Africa” and “Americas” regions have the highest incidence of ADD (about 3.3 and 3.2 episodes per child year, resp.), while Southeast Asia and Africa have the highest burden of severe ADD and diarrhearelated mortality [2, 7]. Infectious agents including viruses, bacteria, and parasites can cause ADD and their prevalence is reported to be different in different regions of the world [8]. Colombia has an estimated incidence of ADD in the range of 3.12 to 6.03 per child year with the highest incidence in infants 6–۱۱ months of age [7]. The etiology of ADD in Colombia had been reported in a few studies; however, most of the studies were small and did not aim to look for all the etiologic agents (e.g., those caused by E. coli pathotypes and caliciviruses). Moreover, the microbiologic detection methods had limited sensitivity [9–۱۲]. The objective of the current study was to determine the feasibility to conduct a large, matched, case-control study to examine the association of viral, bacterial, and parasitic enteropathogens with ADD in children of 2 weeks to 5 years of age in Colombia using advanced microbiologic and molecular techniques. Our work describes the methodology used in the study and results from pilot data. Such detailed studies, aimed at understanding the etiology of ADD, are important to guide appropriate future treatment and public health preventative interventions.