In genes related to drug pharmacokinetics, molecular variations determine interindividual variability in the therapeutic efficacy and adverse drug reactions. The assessment of single-nucleotide variants (SNVs) is used with growing frequency in pharmacogenetic practice, and recently, high-throughput genomic analyses obtained through next-generation sequencing (NGS) have been recognized as powerful tools to identify common, rare and novel variants. These genetic profiles remain underexplored in Latin-American populations, including Colombia. In this study, we investigated the variability of 35 genes included in the ADME core panel (absorption, distribution, metabolism, and excretion) by whole-exome sequencing (WES) of 509 unrelated Colombian individuals with no previous reports of adverse drug reactions. Rare variants were filtered according to the minor allele frequencies (MAF) <1% and potential deleterious consequences. The functional impact of novel and rare missense variants was assessed using an optimized framework for pharmacogenetic variants. Bioinformatic analyses included the identification of clinically validated variants described in PharmGKB and ClinVar databases. Ancestry from WES data was inferred using the R package EthSEQ v2.1.4. Allelic frequencies were compared to other populations reported in the public gnomAD database. Our analysis revealed that rare missense pharmacogenetic variants were 2.1 times more frequent than common variants with 121 variants predicted as potentially deleterious. Rare loss of function (LoF) variants were identified in 65.7% of evaluated genes. Regarding variants with clinical pharmacogenetic effect, our study revealed 89 sequence variations in 28 genes represented by missense (62%), synonymous (22.5%), splice site (11.2%), and indels (3.4%). In this group, ABCB1, ABCC2, CY2B6, CYP2D6, DPYD, NAT2, SLC22A1, and UGTB2B7, are the most polymorphic genes. NAT2, CYP2B6 and DPYD metabolizer phenotypes demonstrated the highest variability. Ancestry analysis indicated admixture in 73% of the population. Allelic frequencies exhibit significant differences with other Latin-American populations, highlighting the importance of pharmacogenomic studies in populations of different ethnicities. Altogether, our data revealed that rare variants are an important source of variability in pharmacogenes involved in the pharmacokinetics of drugs and likely account for the unexplained interindividual variability in drug response. These findings provide evidence of the utility of WES for pharmacogenomic testing and into clinical practice.