Biological therapies are currently used for some diseases which include: autoimmune diseases, cancer, osteoporosis among others; These treatments are long term and costly for health systems, so it is important to know the economic impact of using these medicines. OBJECTIVES:  to estimate the direct costs associated with the use of biological therapies in the health care of patients insured with a health manteinance organization (HMO) in Colombia during the period 2014-2016. METHODS:  A retrospective cross-sectional study was conducted in a cohort of 2597 patients distributed in 7 pathologies (rheumatoid arthritis, autoimmune diseases, cancer, osteoporosis, ophthalmologic, skin and renal pathologies) in 16 cities nationwide, affiliated with a HMO, for a period from January 2014 to August 2016. Total and average costs for the pathology, therapy, city and patient were estimated. Costs were adjusted for inflation and expressed in 2017 US dollars. RESULTS:  The total annual cost of using biological therapies for 2014 was: $ 10,128,248 for 2015: $ 12,844,339 and for 2016 (until August): $ 9,290,887. The most costly cities were Bogota with 27.7% of the total cost followed by Medellín with 15.0%. The most costly pathology was rheumatoid arthritis with 36.5% of the total cost. The average monthly cost per patient for rheumatoid arthritis was $ 594. Cancer: $ 1,545. Autoimmune diseases: $ 683 and ophthalmologic pathologies: $ 431. The therapy with the highest monthly cost was adalimumab with $ 366,224 followed by etanercept with $ 353,496. CONCLUSIONS:  In general, rheumatoid arthritis is the most resource-consuming pathology, however, the average cost per patient of cancer patients is three times higher than patients with rheumatoid arthritis. In addition, although the target population has been increasing, the average costs of using biological therapies have remained stable over this period of time.