Pre-treatment DPYD genotyping of a panel of 4-single nucleotide polymorphism (SNP) including DPYD*2A, DPYD*13, c.2846A$>$T and c.1236A$>$G-HapB3, has been strongly recommended by the current pharmacogenetics guidelines in order to avoid severe fluoropyrimidine (FL)-related toxicity. However, translation to clinical practice is still lagging behind. This requires a better definition of the relationship between genetic variants of DPYD and dose-limiting toxicities (DLTs) and development of new methods to investigate the effect of DPYD variants, such as the DPYD activity score. The aim of the current study was to support the clinical implementation of DPYD genetic testing, by assessing the relationship between DPYD variants in the 4-SNP panel and the risk to develop DLTs and by stratifying patients according to the DPYD gene activity score (GAS) model. (GAS $=$ 1.0 if carriers of one DPYD*2A or DPYD*13 alleles and GAS $=$ 1.5, if carriers of one c.2846A$>$T or c.1236G$>$A-HapB3 allele. Non-carriers GAS $=$ 2.0). A retrospective population of 763 colorectal cancer patients treated with FL-based chemotherapy, was selected and genotyped$.$ Patients carrying at least one decreased function DPYD variant in the 4-SNP panel, displayed a significant association with the risk of developing DLT ($i.e.$ grade $\geq $ 3 non-hematological toxicity or grade $\geq $ 4 hematological toxicity) either within the first three cycles of chemotherapy (OR$=$ 2.7, 95% CI $=$ 1.33-5.41) or during the entire course of treatment (OR $=$ 2.7, 95% CI $=$ 1.42-5.04). Patients' GAS was found to better define the risk of DLT for both acute (GAS $=$ 1.5, OR $=$ 1.80, 95% CI $=$ 0.78-4.15 and GAS $=$ 1.0, OR $=$ 10.12, 95% CI $=$ 2.55-40.20) and total toxicity (GAS $=$ 1.5, OR $=$ 2.08, 95% CI $=$ 1.02-4.27 and GAS $=$ 1, OR $=$ 7.09, 95% CI $=$ 1.69-29.65). In conclusion, the present study demonstrates that the DPYD 4-SNP panel and the associated GAS can predict the occurrence of DLT related to treatment with FL. These findings further support the implementation of pre-emptive DPYD genotyping in the routine clinical practice.